NZ615735A - Cd37-binding molecules and immunoconjugates thereof - Google Patents

Abstract

Discloses the use of a purified humanized antibody or antigen-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for treating a patient having an autoimmune or inflammatory disease, wherein said humanized antibody or fragment thereof retains the ability of at least its chimeric or murine parent antibody to induce apoptosis in vitro in the absence of a cross-linking agent.

Description

/031648 CD37-BINDING MOLECULES AND IMMUNOCONJUGATES F Field of the Inventior’: The field of the invention generally relates to antibodies, antigen-binding fragments thereof, polypeptides, and immunoconjugates that bind to CD37, as well as to methods of using such CD37— binding molecules for the treatment of diseases, such as autoimmune diseases and inflammatory diseases.

Background of the Invention Leukocyte n CD37 ("CD37"), also known as GP52—40, tetraspanin—26, or TSPAN26, is a transmembrane protein of the tetraspanin superfamily (Maecker et al., 1997 FASEB J. 112428-442). It is a heavily glycosylated protein with four transmembrane domains that is expressed on B cells during the pre-B to peripheral mature B-cell stages, but is reportedly absent on terminal differentiation to plasma cells. (Link et al., 1987, J Pathol. 152:12—21). The CD37 n is only weakly expressed on T-cells, d cells, and granulocytes (Schwartz-Albiez et al. 1988, J. Immunol., 905-914). However, CD37 is also expressed on malignant B-cells such as tleose founding non-Hodgkin’s lymphoma (NHL) and c lymphoid leukemia (CLL) (Moore et al. 1986, J Immunol. 137(9):3013-8).

While the exact logical role of CD37 is unclear, studies in CD37-deficient mice suggest an immunoregulatory function. Although mice deficient in CD37 expression have normal development (Knobeloch et al. 2000, Mol Cell Biol., 20(15):5363—9), in the C57/Bl6 background, CD'37-/- T cells are hyper-proliferative (van Spriel et al., J Immunol. 172, 2953 (2004)), CD37-/- dendritic cells (DC) exhibit an increased antigen presentation (Sheng et al., Eur J Immunol. 39, 50 (2009)), and CD3 7—/— macrophages show increased -l—induced IL—6 tion (Meyer-Wentrup et al., J Immunol. 178, 154 (2007)).

CD3 7-deficient C57/Bl6 mice also contain significantly higher level of IgA than the wild—type mice (van Spriel et al., PLoS Pathol. 5, e1000338 (2009) and Rops et al., Am J Pathol. 176, 2188 (2010)). All of these results suggest a general regulatory role of CD37 in the immune system. Interestingly, crosslinking of CD37 antigen by antibody on human T cells inhibits T cell proliferation d by CD3 stimulation (van Spriel eta1., J Immunol. 172, 2953 (2004)).

Antibodies are emerging as a promising method to treat human diseases including autoimmune es. Cureently, an anti-CD20 antibody called rituximab has been approved for toid arthritis (RA) treatment (Edwards JC et al. 2006, Nat Rev Immunol. 6: 119), Rituximab is used in the United States in combination with methotrexate (MTX) to reduce signs and ms in adult patients with moderately— to severely-active RA who have had an inadequate response to at least one TNF antagonist. Many studies s the use of rét’uximab in a variety of non—malignant autoimmune or inflammatory disorders, including RA, in which B-cells and autoantibodies appear to play a role in disease pathophysiology. Edwards et al., Biochem Soc. Trans. 30:824—828 (2002). Targeting of CD20 W0 2012/135740 PCT/U82012/031648 _ 2 _ using anti-CD20 antibody has been reported to potentially relieve signs and symptoms of a number of autoimmune or inflammatory diseases including, for example, RA ro et al., Ann. Rheum. Dis. 61 :883-888 (2002); Edwards et al., Arthritis Rheum, 46 (Suppl. 9): S46 (2002); Stahl et al., Ann. Rheum.

Dis., 62 (Suppl. 1): OP004 (2003); Emery et al., Arthritis Rheum. 48(9): S439 (2003)), lupus (Eisenberg, Arthritis. Res. Ther. 5:157-159 (2003); Leandro et al. Arthritis Rheum. 46: 2673—2677 (2002); Goranan et al., Lupus, 13: 312—316 (2004)), immune thrombocytopenic purpura (D'Arena et al., Leuk. Lymphoma 44:561-562 (2003); Stasi et al., Blood, 98: 952-957 (2001); Saleh et al., Semin. Oncol., 27 (Supp 12):99— 103 (2000); Zaja et al., Haematologica, 87:189-195 (2002); Ratanatharathom et al., Ann. Int. Med., 133:275-279 (2000)), pure red cell aplasia (Auner et al., Br. J. Haematol, 116:725-728 (2002)); autoimmune anemia (Zaja et al., supra um s in Haematologica 87:3 36 (2002)), cold agglutinin e (Layios et al., Leukemia, 15:187—8 (2001); Berentsen et al., Blood, 103: 928 (2004); Berentsen et al., Br. J. Haematol., 115:79-83 (2001); r, Br. J. ol, 112:1083-1090 (2001); Zaja et al., Br. J. Haematol, 115:232-233 (2001)), type B me of severe insulin resistance (Coll et al., N. Engl. J. Med., 350:310-311 (2004), mixed cryoglobulinerrnia (DeVita et al., Arthrétis Rheum. 46 Suppl. 91S206/S469 ), myasthenia gravis (Zaja et al., Neurology, 55:1062-1063 (2000); Wylam et al., J. Pediatr., 143:674—677 (2003)), Wegener’s granulomatosis (Specks et al., Arthritis & Rheumatism 44:2836—2840 ), microscopic polyangiitis (MPA), refractory gus vulgaris (Dupuy et al., Arch Dermatol, 140:91—96 (2004)), dermatomyositis (Levine, Arthritis Rheum, 46 (Suppl. 9)281299 (2002)), Sjogren's syndrome (Somer et al., Arthritis & Rheumatism, 49:394—398 (2003)), active type-II mixed cryoglobulinemia (Zaja et al., Blood, 101:3 34 ), pemphigus vulgaris (Dupay et al., Arch. Dermatol, 140:91—95 (2004)), autoimmune neuropathy onk et al., J. Neurol. Neurosurg.

Psychiatry 74:485-489 (2003)), paraneoplastic opsoclonus—myoclonus syndrome atelli et al.

Neurology 60 (Suppl. 1) POS.128:A395 (2003)), and relapsing—remitting multiple sclerosis (RRMS).

Cross et a1. (abstract) "Preliminary Results from a Phase II Trial of Rituximab in MS” Eighth Annual Meeting of the Americas Committees for Research and Treatment in le Sclerosis, 20-21 (2003).

In animal models, B—cell depletion using antibodies against B—cell antigens such as CD20 has been shown to inhibit or ameliorate several autoimmune es including ic lupus erytléematosus (SLE), mental autoimmune encephalomyelitis (EAE; mouse model of multiple sclerosis), type-1 diabetes (TlD) and rheumatoid arthritis (RA). Rituximab has been shown to deplete both malignant and normal B cells in vivo in animal models as well as patients {Malone}; DG et al, Blood. l994;84(8):2457~66; Reff ME, et al. Blood. 1994;83(2):435-45; Schroder C, et at. Transpl lmmunol. 2003;12(l):l9—28). It can aiso deplete nermal B»cells from human peripheral blood rnononnclear celis ) in in with; experiments (Vugmeyster Y, et a1, Cytometry A. 2003;52(2):101—9; Vngmeyster Y and Hewell Kint lmrntmopharmacol. 2004;4(8):l l l7~24).

W0 2012/135740 2012/031648 _ 3 _ Campath-lH uzumab), an anti-CD52 ic IgGl, binds to the CD52 n, which is highly expressed on all lymphocytes (Ginaldi L, et al,Leuk Res. 1998 Feb;22(2):l85-91; Hale G, et al, Tissue Antigens. 1990 (3):l 1 8-27). It is used in patients to deplete malignant lymphocytes and is approved for treating c lymphocytic leukemia. It has also shown efficacy in treating multiple sclerosis and is currently in Phase III clinical testing (N Engl J Med 2008; 359:1786-1801; ClinicalTrials.gov NCT00530348 & 48405). It has been shown to deplete normal lymphocytes in vitro as well (Hale G, et al. Blood. 1983 Oct;62(4):873—82; Waldmann H and Hale G Philos Trans R Soc Lond B Biol Sci. 2005 Sep 29;360(1461):1707-1l).

CD37-binding agents are also being tested as potential therapeutics for B-cell malignancies.

Emergent Biosolutions (formerly Trubion ceuticals) developed the CD37-binding agents SMIP- 016 and TRU-016 (Zhao et al., 2007, Blood, 110:2569—2577). SMIP—016 is a single chain polypeptide that includes variable regions from a hybridoma and engineered human constant regions. TRU-016 is a humanized version of the anti-CD37 SMIP protein. See e.g. U.S. Published Application No. 2007/0059306. TRU—Ol6 is being tested clinically for the ent of chronic lymphocytic ia (CLL). Boehringer Ingelheim has also disclosed a CD37 binding agent in International Published Application No. . However, no CDC activity has been described for any of these binding agents and no in vitro pro-apoptotic ty has been described in the absence of cross-linking Radio—immunotherapy (RIT) has been attempted using a radio—labeled anti-CD37 antibody MB—l in two separate trials. Therapeutic doses of 131I-MB-l were stered to six relapsed NHL patients (Press et al. 1989 J Clin Oncol. 7(8):1027—38; Press at el. 1993, N Engl J Med. 329(17):l2l9-24).

All six patients achieved a complete remission (CR) with a duration of four to thirty—one months. In another trial, 131I-MB—l was administered to ten relapsed NHL patients (Kaminski et al. 1992 J Clin Oncol. :l696-7ll). A total of four patients had a response ranging in duration from two to six months, although only one CR was reported. However, not all ts could be treated due to an unfavorable biodistribution of the radio—label which raised concern for radiation exposure of vital non- target organs. Indeed, RIT related toxicities were observed in these trials including severe myelosupression and cardiopulmonary toxicity. While these clinical data suggest that anti—CD37 radio- immunoconjugates may be ive, these therapies are some to administer, and at relapse post- RIT patients cannot be retreated with RIT due to the risks associated with high doses of radiation.

To overcome the limitations of RIT, antibody—cytotoxic agent conjugates (ACC), also called antibody-drug conjugates (ADC), have been ped. These are immunoconjugates that include a cytotoxic agent covalently linked to an antibody through a chemical linker which can allow for specific delivery of cytotoxic drugs to cells expressing a protein recognized by the antibody. However, proteins that are poorly internalized are not considered to be favorable targets for such therapeutics. CD37 is W0 2012/135740 PCT/U82012/031648 _ 4 - structurally similar to CD20 as both antigens contain four transmembrane domains, although CD20 is not part of the tetraspanin family (Tedder et a1. 1989, J. Immun. 142: 2560—2568). Antibodies against several B-cell ns including CD37 and CD20 have been studied for their ability to undergo endocytosis and degradation (Press et al. 1989, Cancer Res. 49(17):4906—12, and Press et a1. 1994, Blood. 83(5):1390-7).

The anti-CD37 antibody MB-l was retained on the cell surface and alized slowly in Daudi lymphoma cells in vitro. The MB—l antibody also had a low rate of endocytosis and ellular metabolism in NHL patient cells in vitro. Similar results were obtained with the anti—CD20 antibody 1F5, which was also ed mainly on the lymphoma cell surface and internalized . ADCs of CD20 antibodies have been studied previously but have not demonstrated significantly strong potency, especially when non-disulfrde or acid stable linkers are used (see for example Polson et al., 2009, Cancer Res, 69(6):2358-2364). In light of these observations, CD37 has not been considered a favorable target for antibody-drug conjugates.

While their role in cancer treatment has been studied, the potential effect of CD37-directed therapies such as antibodies, antibody derivatives or immunoconjugates on cells involved in autoimmune diseases, inflammatory diseases or other disorders of the immune system is not well understood. Furthermore, none of the compounds bed above have been demonstrated to induce depletion of target cells involved in manifestation or progression of these types of diseases.

Therefore, there exists a need for CD37 binding agents including antibodies, antigen-binding fragments thereof, and antibody-drug conjugates oconjugates) as a means to treat mune diseases, inflammatory diseases, or other disorders of the immune system. The present ion addresses that need.

BRIEF SUMMARY OF THE INVENTION [0012| In one aspect, the present disclosure provides a method for depleting s or treating a disease associated with aberrant B-cell activity, comprising administering to a patient an effective amount of a humanized CD37 targeting antibody or immunoconjugate provided herein. In some embodiments, the B—cells are non-cancerous lls. In some embodiments, the B—cells do not press CD37.

In certain embodiments, the disease associated with aberrant B-cell activity is a disease associated with B-cell autoantibody production, and/or a disease associated with inappropriate T-cell stimulation in connection with a B—cell pathway.

In certain embodiments, the disease characterized by autoantibody production is rheumatoid arthritis, multiple sclerosis, type I diabetes us, idiopathic inflammatory myopathy, systemic lupus erythematosus (SLE), enia gravis, Grave's disease, derrnatomyositis, polymyositis, or other autoimmune diseases, W0 35740 PCT/U82012/031648 _ 5 _ In certain embodiments, the t disclosure provides a method for depleting a B—cell sing contacting a B-cell (e.g., in a population of cells comprising a non-cancerous B—cell) with an antibody or antigen binding fragment thereof that specifically binds to CD37, n the antibody or fragment thereof is capable of inducing apoptosis in vitro in the e of a cross—linking agent. In certain ments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD37, wherein the antibody or nt thereof is capable of inducing apoptosis in vitro in the e of a cross—linking agent. In some embodiments, the antibody or antigen—binding fragment thereof is also capable of inducing complement dependent cytotoxicity (CDC). In some embodiments, the antibody or n- binding nt thereof is also capable of inducing antibody dependent cell mediated cytotoxicity (ADCC). In some embodiments, the antibody or antigen-binding fragment thereof has a long serum half- life.

In certain embodiments, the present disclosure provides a method for depleting a B-cell comprising ting a B-cell (e.g., in a population of cells comprising a non-cancerous B-cell) with an antibody or antigen binding fragment f that specifically binds to the same CD37 epitope as an antibody selected from the group consisting of: (a) an antibody comprising the polypeptide of SEQ ID X0255 and the polypeptide of SEQ ID NO:72; (b) an antibody comprising the polypeptide of SEQ ID NO:56 and the polypeptide of SEQ ID NO:73; (c) an antibody comprising the polypeptide of SEQ ID \10257 and the polypeptide of SEQ ID NO:74; (d) an antibody comprising the polypeptide of SEQ ID \O:58 and the polypeptide of SEQ ID NO:74; (e) an dy comprising the polypeptide of SEQ ID I\O:59 and the polypeptide of SEQ ID NO:75; (i) an antibody comprising the ptide of SEQ ID \'O:60 and the polypeptide of SEQ ID NO:76; (g) an antibody comprising the polypeptide of SEQ ID N026] and the polypeptide of SEQ ID NO:77; (h) an antibody sing the polypeptide of SEQ ID \'O:62 and the polypeptide of SEQ ID NO:78; (i) an antibody comprising the polypeptide of SEQ ID \IO:63 and the polypeptide of SEQ ID NO:79; (j) an antibody comprising the polypeptide of SEQ ID \‘Oz64 and the polypeptide of SEQ ID NO:80; (k) an antibody sing the polypeptide of SEQ ID XO:65 and the polypeptide of SEQ ID NO:81; (1) an antibody comprising the polypeptide of SEQ ID ROz66 and the ptide of SEQ ID NO:82; (m) an antibody comprising the polypeptide of SEQ ID V067 and the polypeptide of SEQ ID NO:83; (n) an antibody comprising the polypeptide of SEQ ID NO:68 and the polypeptide of SEQ ID NOz84; (0) an antibody comprising the polypeptide of SEQ ID N069 and the polypeptide of SEQ ID NO:85; (p) an antibody comprising the polypeptide of SEQ ID I\O:70 and the ptide of SEQ ID N0286; (q) an antibody comprising the polypeptide of SEQ ID NO:71 and the polypeptide of SEQ ID NO:87; and (r) an antibody comprising the polypeptide of SEQ ID NO:177 and the polypeptide of SEQ ID NO:l78,.

W0 35740 PCT/U82012/031648 _ 6 _ In certain ments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease sing administering to the patient a therapeutically ive amount of an antibody or antigen-binding fragment thereof that specifically binds to the same CD37 epitope as an antibody selected from the group described above. In some embodiments, the antibody or n-binding fragment thereof competitively inhibits an antibody selected from the group bed above.

In certain embodiments, the present disclosure es a method for depleting a B-cell comprising contacting a B—cell (e.g., in a population of cells comprising a non-cancerous B—cell) with an antibody or antigen-binding fragment thereof that specifically binds to CD37 and specifically binds to the ptide of SEQ ID NO: 184. In certain embodiments, the present disclosure provides a method for treating a patient having an autoimmune or atory disease comprising administering to the patient a therapeutically effective amount of an antibody or antigen—binding fragment thereof that specifically binds to CD37 and specifically binds to the polypeptide of SEQ ID NO: 184. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to the polypeptide of SEQ ID NO: 185.

In certain embodiments, the t disclosure provides a method for depleting a B-cell comprising contacting a B—cell (e.g., in a population of cells sing a non—cancerous B-cell) with an antibody or antigen-binding fragment thereof that specifically binds to CD37 and does not specifically bind to the polypeptide of SEQ ID NO: 185. In certain embodiments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD37 and does not specifically bind to the polypeptide of SEQ ID NO: 185.

In certain ments, the present disclosure provides a method for depleting a B-cell comprising contacting a B-cell (e.g., in a population of cells comprising a non-cancerous ) with an antibody or antigen-binding fragment thereof produced by a oma selected from the group consisting of ATCC Deposit ation PTA-10664, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10665, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10666, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation 667, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10668, deposited with the ATCC on February 18, 2010, ATCC t Designation PTA-10669, deposited with the ATCC on February 18, 2010, and ATCC Deposit Designation PTA—10670, deposited with the ATCC on ry 18, 2010. In n embodiments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a therapeutically ive amount of an antibody or antigen-binding fragment thereof produced by a hybridoma described above...

W0 2012/135740 PCT/U82012/031648 - 7 _ In certain embodiments, the present disclosure provides a method for depleting a B-cell comprising contacting a B-cell (e.g., in a population of cells comprising a non—cancerous B-cell) with an antibody or antigen—binding fragment f that specifically binds to CD37, wherein the antibody comprises polypeptide sequences selected from the group consisting of: (a) SEQ ID NOs: 4, 5, and 6 and SEQ ID NOS: 28, 29, and 30; (b) SEQ ID NOS: 7, 8, and 9 and SEQ ID NOS: 31, 32, and 33; (c) SEQ ID NOS: 10, 11, and 12 and SEQ ID NOS: 34, 35, and 36; (d) SEQ ID NOS: 13, 14, and 15 and SEQ ID NOS: 37, 38, and 39; (e) SEQ ID NOS: 13, 14, and 15 and SEQ ID NOS: 37, 40, and 39; (f) SEQ ID NOS: 16, 17, and 18 and SEQ ID NOS: 41, 42, and 43; (g) SEQ ID N05: 19, 20, and 21 and SEQ ID NOS: 44, 45, and 46; (h) SEQ ID NOS: 19, 20, and 21 and SEQ ID NOS: 44, 47, and 46; (i) SEQ ID NOS; 22, 23, and 24 and SEQ ID NOS: 48, 49, and 50; (j) SEQ ID NOS: 22, 23, and 24 and SEQ ID NOs: 48, 51, and 50; (k) SEQ ID NOS: 25, 26, and 27 and SEQ ID NOS: 52, 53, and 54; (l) SEQ ID N05: 171, 172 or 181, and 173 and SEQ ID NOS: 174, 175, and 176; (m) variants of (a) to (1) comprising 1, 2, 3, or 4 conservative amino acid substitutions. In certain embodiments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof with an antibody or antigen-binding fragment thereof that Specifically binds to CD37, wherein the antibody ses polypeptide sequences ed from the group described above. In some embodiments, the antibody or antigen-binding fragment f ses polypeptide sequences that are at least 90% identical to polypeptide sequences described above. In some embodiments, the polypeptide sequences are at least 95% identical to the polypeptide sequences. In some ments, the polypeptide sequences are at least 99% identical to the polypeptide sequences. In some ments, the antibody or antigen-binding fragment thereof comprises polypeptide sequences that are at least 90% identical, at least 95% identical, at least 99% identical, or idential to the polypeptide sequences of SEQ ID NO: 57 and SEQ ID NO:74. In some embodiments, the antibody or antigen—binding fragment thereof comprises ptide sequences that are at least 90% identical, at least 95% cal, at least 99% identical, or idential to the polypeptide sequences of SEQ ID NO: 58 and SEQ ID NO:74. In some embodiments, the antibody or n-binding nt thereof comprises polypeptide sequences that are at least 90% identical, at least 95% identical, at least 99% cal, or idential to the polypeptide sequences of SEQ ID NO: 63 and SEQ ID NO:79. In some ments, the antibody or antigen-binding fragment thereof comprises polypeptide sequences that are at least 90% cal, at least 95% identical, at least 99% identical, or idential to the ptide sequences of SEQ ID NO: 65 and SEQ ID NO:81.

In some embodiments, the antibody or antigen binding fragment thereof is murine, non- human, humanized, chimeric, aced, or human.

In some embodiments, the antibody or antibody fragment is capable of inducing apoptosis of a cell expressing CD37 in vitro in the absence of cross-linking agents. In some embodiments, the antibody W0 2012/135740 PCT/U82012/031648 _ g _ or antigen g fragment is capable of inducing complement dependent cytotoxicity (CDC). In some ments, the antibody is e of inducing antibody dependent cell ed cytotoxicity (ADCC).

In certain ments, the present disclosure provides a method for depleting a B-cell comprising contacting a B-cell (e.g., in a tion of cells comprising a non-cancerous B-cell) with a human or humanized antibody or n binding fragment thereof that specifically binds to CD37, wherein the antibody or nt thereof is capable of inducing apoptosis of a cell sing CD37 in vitro in the absence of cross—linking agents. In certain embodiments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising stering to the patient a therapeutically effective amount of a human or humanized antibody or antigen binding fragment f that specifically binds to CD37, wherein the antibody or fragment thereof is capable of inducing apoptosis of a cell sing CD37 in vitro in the absence of cross-linking agents. In some embodiments, the human or humanized antibody or antigen binding fragment thereof is also capable of inducing complement dependent cytotoxicity (CDC). In some embodiments, the human or humanized antibody or antigen binding fragment thereof is also capable of inducing antibody dependent cell mediated cytotoxicity (ADCC).

In some embodiments, the dy or antigen-binding fragment binds to human CD37 and macaque CD37.

In some embodiments, the dy is a full length antibody. In some embodiments, an antigen-binding fragment is used. In some embodiments, the antibody or antigen—binding nt thereof comprises a Fab, Fab', F(ab')2, F(1, single chain Fv or scFv, disulfide linked Fv, V-NAR , IgNar, intrabody, IgGACHZ, minibody, F(ab')3, ody, triabody, diabody, single-domain antibody, DVD—lg, Fcab, mAb2, (scFv)2, or scFv-Fc.

In some embodiments, the antibody or antigen-binding fragment thereof is linked via a linker (L) to a cytotoxic agent (C) to form an immunoconjugate.

In certain embodiments, the present disclosure provides a method for depleting a B—cell comprising contacting a B-cell (e.g., in a population of cells comprising a non-cancerous B-cell) with a composition comprising an immunoconjugate having the formula (A) - (L) - (C), wherein: (A) is an dy or antigen binding fragment that specifically binds to CD37; (L) is a non-cleavable linker; and (C) is a cytotoxic agent; and wherein the linker (L) links (A) to (C). In certain embodiments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a therapeutically effective amount of a composition comprising an immunoconjugate having the a (A) — (L) - (C), wherein: (A) is an antibody or antigen binding fragment that specifically binds to CD37; (L) is a non-cleavable ; and (C) is a cytotoxic agent; and wherein the linker (L) links (A) to (C). In some embodiments, the immunoconjugate has a serum half—life that is comparable to that of the naked antibody, W0 2012/135740 PCT/U82012/031648 In certain embodiments, the present disclosure provides a method for depleting a B-cell comprising contacting a B-cell (e.g., in a population of cells comprising a non-cancerous B-cell) with a composition comprising an immunoconjugate having the formula (A) - (L) — (C), wherein: (A) is an antibody or antigen binding nt that specifically binds to CD37; (L) is a linker; and (C) is a maytansinoid; and n the linker (L) links (A) to (C). In certain embodiments, the present disclosure provides a method for treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a eutically effective amount of a composition comprising an conjugate having the a (A) - (L) - (C), wherein: (A) is an antibody or antigen binding fragment that specifically binds to CD37; (L) is a linker; and (C) is a maytansinoid; and wherein the linker (L) links (A) to (C).

In some embodiments, the linker is a non-cleavable linker. In some embodiments, the immunoconjugate further comprises a second (C). In some embodiments, the immunoconjugate further comprises a third (C). In some embodiments, the immunoconjugate further comprises a fourth (C). In some embodiments, the immunoconjugate ses 2—6 (C). In some embodiments, the immunoconjugate comprises 3-4 (C).

In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non—cleavable linker, a hydrophilic linker, and a dicarboxylic acid based linker. In some embodiments, the linker is selected from the group consisting of: N—succinimidyl 4-(2—pyridyldithio)pentanoate (SPP); N—succinimidyl 4-(2—pyridyldithio)butanoate (SPDB) or N-succinimidyl 4-(2-pyridyldithio)—2- sulfobutanoate (sulfo-SPDB); N-succinimidyl 4—(maleirnidomethyl) cyclohexanecarboxylate (SMCC); N- sulfosuccinimidyl 4-(maleimidomethyl) exanecarboxylate (sulfoSMCC); N—succinimidyl (iodoacetyl)—aminobenzoate (SIAB); and N—succinimidyl—[(N-maleimidopropionamido)— thyleneglycol] ester (NHS-PEG4-maleimide). In some embodiments, the linker is N—succinimidyl— [(N-maleimidopropionamido)—tetraethyleneglycol] ester (NHS-PEG4-maleimide).

In some embodiments, the cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, doxornbicin, a modified bicin, iazepine, taxoid, CC— 1065, CC-lO65 analog, duocarznycin, duocarznycin analog, eamicin, dolastatin, atin analog, aristatin, tomaymycin derivative, and leptomycin derivative or a prodrug of the agent. In some embodiments, the cytotoxic agent is a maytansinoid. In some ments, the xic agent is N(2')— deacetyl-N(2')-(3-mercapto—l-oxopropyl)—maytansine (DMl) or N(2')—deacetyl-N2—(4-mercapto—4-methyl— l-oxopentyl)—maytansine (DM4).

In some embodiments, the composition comprising an conjugate comprises multiple cytotoxic agents (C) with an e of about 3 to about 4 (C) per (A). In some embodiments, the irrirnunoeonjugates have an average of about 3.5 (C) per (A). In some embodiments, the immunoccnjugates have an average ofabout 3.5 i 0.5 (C) per (A).

WO 35740 _ 10 -.

In some embodiments, the composition comprising an immunoconjugate comprises an antibody comprising SEQ ID NO:57 and SEQ ID NO:74 or SEQ ID NO:58 and SEQ ID NO:74, an SMCC linker, and DM1. In some ments, the composition comprising an immunoconjugate ses an antibody comprising SEQ ID NO:63 and SEQ ID NO:79, an SMCC , and DM1. In some embodiments, the composition comprising an conjugate comprises an antibody comprising SEQ ID NO:65 and SEQ ID NO:81, an SMCC linker, and DM1.

In some embodiments, the antibody or antigen-binding fragment is capable of depleting B- cells. In some embodiments, the dy or antigen—binding fragment is capable of inhibiting T-cell responses.

In some embodiments, the B-cell is in a composition further comprising a T-cell. In some embodiments, the B—cell is in a composition comprising peripheral blood mononuclear cells. In some embodiments, the peripheral blood mononuclear cells were obtained from a human. In some ments, the B—cell is in whole blood. In some embodiments, the whole blood was obtained from a human. In some embodiments, the B—cell is in an organism. In some embodiments, the B-cell is in a patient having an autoimmune or inflammatory disease.

In some embodiments, the B-cell is an autoreactive B—cell.

In some embodiments, at least about 30% of B-cells are depleted. In some ments, less than about 5% of s are depleted.

In some embodiments, a second therapeutic agent is administered. In some embodiments, the second therapeutic is selected from the group consisting of methotrexate, an anti—CD20 therapeutic, an anti-IL-6 receptor therapeutic, ar L—12/23p40 eutic, a chemotherapeutic, an immunosuppressant, an anti-Interferon beta-1a therapeutic, glatiramer e, an anti-0L4—integrin eutic, fingolimod, an anti-BLys therapeutic, CTLA-Fc, or an anti-TNF therapeutic. In some embodiments, the second therapeutic is an dy directed against an antigen selected from a group consisting of CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD3 8, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138, and CD152. In some embodiments, the second therapeutic is an antibody directed against an antigen selected from the group consisting of 1L-2, IL—6, IL—12, 1L-23, IL—12/23 p40, IL—17, IFNy, TNFoc, IFNOL, IL—15, IL-21, IL—Ia, 1L- lb, IL—18, IL-8, IL—4, GM—CSF, IL—3, and IL—5.

In some embodiments, the autoimmune or inflammatory disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type I diabetes mellitas, idiopathic atory myopathy, systemic lupus erythematosus (SLE), myasthenia gravis, Grave's disease, dermatomyositis, polymyositis, Crohn’s diasease, ulcerative colitis, gastritis, Hashimoto’s ditis, asthma, psoriasis, psoriatic arthritis, dertmatitis, systemic sclerodema and sclerosis, inflammatory bowel disease (IBD), respiratory distress syndrome, meningitis, encephalitis, uveitis, glmerulonephritis, eczema, atherosclerosis, leukocyte adhesion deficiency, Raynaud’s syndrome, Sjögren’s syndrome, Reiter’s disease, Beheet’s disease,immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias, acute idiopathic ocytopenic purpura, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, enia , lupus nephritis, atopic dermatitis, pemphigus vulgaris, opsoclonus-myoclonus syndrome, pure red cell aplasia, mixed cryoglobulinemia, sing spondylitis, hepatitis ciated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous goid, hemophilia A, noproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves’ dysthyroid disease, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss me, juvenile onset diabetes, hemolytic anemia, atopic dermatitis, systemic sclerosis, Sjögren’s syndrome and ulonephritis, dermatomyositis, anti-neutrophil cytoplasmic antibody (ANCA), aplastic anemia, autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman's me, Goodpasture's syndrome, solid organ transplant rejection, graft versus host disease (GVHD), autoimmune hepatitis, lymphoid interstitial nitis, HIV, bronchiolitis obliterans (non-transplant), Guillain-Barre Syndrome, large vessel vasculitis, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's Disease, polyarteritis nodosa, Wegener’s granulomatosis, microscopic giitis (MPA), Omenn’s syndrome, chronic renal failure, acute infectious mononucleosis, HIV and herpes virus associated diseases. [0039a] Definitions of the specific embodiments of the invention as claimed herein follow. [0039b] According to a first embodiment of the invention, there is provided use of a ed humanized antibody or antigen-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for treating a patient having an autoimmune or inflammatory disease, wherein said zed antibody or nt thereof retains the ability of at least its chimeric or murine parent antibody to induce apoptosis in vitro in the absence of a cross-linking agent. [0039c] According to a second embodiment of the ion, there is provided use of a purified zed antibody or antigen-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for ing a B-cell in a population of cells comprising a noncancerous , wherein said humanized antibody or fragment thereof retains the ability of at least its chimeric or murine parent antibody to induce apoptosis in vitro in the e of a crosslinking agent. . - 11a - [0039d] According to a third embodiment of the invention, there is provided use of a purified zed antibody or antigen-binding fragment thereof that specifically binds to CD37 in the preparation of a ment for treating a patient having an mune or inflammatory disease, wherein the antibody or antigen-binding fragment thereof comprises polypeptide sequences selected from the group consisting of: (a) SEQ ID NO:57 and SEQ ID NO:74; (b) SEQ ID NO:58 and SEQ ID NO:74; (c) SEQ ID NO:63 and SEQ ID NO:79; (d) SEQ ID NO:65 and SEQ ID NO:81; (e) SEQ ID NO:67 and SEQ ID NO:83; (f) SEQ ID NO:69 and SEQ ID NO:85; and (g) SEQ ID NO:71 and SEQ ID NO:87. [0039e] According to a fourth embodiment of the invention, there is provided use of a purified humanized antibody or antigen-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for depleting a B-cell in a population of cells comprising a noncancerous B-cell, wherein the antibody or antigen-binding fragment thereof comprises polypeptide sequences ed from the group consisting of: (a) SEQ ID NO:57 and SEQ ID NO:74; (b) SEQ ID NO:58 and SEQ ID NO:74; (c) SEQ ID NO:63 and SEQ ID NO:79; (d) SEQ ID NO:65 and SEQ ID NO:81; (e) SEQ ID NO:67 and SEQ ID NO:83; (f) SEQ ID NO:69 and SEQ ID NO:85; and (g) SEQ ID NO:71 and SEQ ID NO:87.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES Figure 1 depicts an FL2-H (PE) histogram y for a flow cytometry experiment with human B-cells. The following ions are shown: antibody control (dark filled), e control stain (light filled), anti-CD37 stain (thick black line), and anti-CD20 stain (dashed line) for - 11b - CD19+ B-cells.

Figure 2 depicts the results of in vitro depletion ments using purified human PBMC samples treated with 10 μg/mL of huCD37-3, huCD37SMCC-DM1, huCD37-50, SMCC-DM1, mab, TRU-016, or alemtuzumab. Results from two different donors are shown in panel A and B.

Figure 3 depicts the results of in vitro depletion experiments using purified human PBMC samples treated with varying concentrations of huCD37SMCC-DM1. Results from two different donors are shown in panels A and B. Figure 3 (C) shows the results using huCD37-3, huCD37-38, huCD37-50 and huCD37-56.

Figure 4 depicts the results of in vitro depletion experiments using unpurified whole human blood samples treated with 10 μg/mL of huCD37-3, SMCC-DM1, huCD37-50, huCD37SMCC-DM1, rituximab, TRU-016, or alemtuzumab.

[TEXT CONTINUES ON PAGE 12] {new Figure 5 depicts the results of in vine depletion experiments using unpnrified whole human blood samples treated with vaming concentrations of (A) hnCDB 7-3, huClIXW—B—Sl‘vfiiCC~li)lVll, and ritnxiinab and (B) huCDBY-IB, huCDW—lSMCC-DMI , huCD37«50, and ritnximab, EQMSE Figure 6 s release of lFN--y (Interferon), "l‘NF~€x (Tumor Necrosis Factor) and Ibo (lnterleukimé) measured by ELlSpot as number of spots per leGEZS peripheral blood mononuclear cells (PBMCS) from one y human donor incubated for ill—20 hours with compounds at a concentration of 2,5 rig/ml;- to 25$ gig/nil... {0046} Figure ‘7 depicts release of 119wa (Interferon), TNF~«:L {Tumor Necrosis Factor) and lire (interleukineé) ed by ELlSpot as mimber of spots per 5x“? peripheral blood mononuclear cells (PBMCs) from a second y human donor incubated for 18-20 hours with compounds at a concentration of 2.5 ng/mL to 250' ug/mL.

Figure 8 depicts the binding curve of anti-muCD37 monoclonal antibody clone 252—3.

Figure 9 shows the activity of the 252—3 antibody in depleting peripheral blood B cells (A) and in inhibiting EAE (B) in C57B1/6 mice. In (A), each symbol represent one mouse; to compare the B cell level in control vs. experimental mice, B cell level was normalized with T cell level and ratio of B/T cell in control mice was considered 100%. In (B), open and closed symbols represent mean of EAE score in control group (n=10) and 252—3 antibody treated group (n=10), respectively; arrow tes day of antibody injection.

Figure 10 shows the ty of the 252-3 dy in depleting peripheral blood B cells (A) and in inhibiting TlD (B) in NOD mice. In (A), each symbol represent one mouse; to compare the B cell level in control vs. experimental mice, B cell level was ized with T cell level and ratio of B/T cell in control mice was considered 100%. In (B), open and closed symbols represent the diabetes incidence in l group (n36) and 252—3 antibody d group (n=6), tively.

Figure 11 shows the activity of the 252-3 antibody in depleting peripheral blood B cells (A) and in inhibiting CIA (B) in DBA/l mice. In (A), each symbol represent one mouse; to compare the B cell level in control vs. experimental mice, B cell level was normalized with T cell level and ratio of B/T cell in control mice was considered 100%. In (B), open and closed symbols represents mean of CIA score in control group (n=l2) and 252-3 antibody treated group (n:12), respectively; arrow indicates day of antibody injection.

DETAILED DESCRIPTION OF THE INVENTION {GoSE} The present invention provides s of depleting B~cells and of treating diseases ated with aberrant Bucell activity and/or aberrant ll stimulation in connection with a B~cell pathway using (2937 binding molecules. _ 13 _ I. Definitions To facilitate an understanding of the t invention, a number of terms and phrases are defined below.

The term CD37 as used herein, refers to any native CD37, unless otherwise indicated. CD37 is also referred to as GP52-40, leukocyte antigen CD37, and Tetraspanin—26. The term "CD37" encompasses "full-length," unprocessed CD37 as well as any form of CD37 that results from processing in the cell. The term also encompasses naturally occurring variants of CD37, e.g., splice variants, allelic variants, and ms. The CD37 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from r source, or prepared by recombinant or synthetic methods.

The term "antibody" means an immunoglobulin le that recognizes and specifically binds to a target, such as a n, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen ition site within the variable region of the immunoglobulin le. As used , the term "antibody" encompasses intact onal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab’, F(ab')2, and Fv fragments), single chain Fv (scFv) s, multispecific antibodies such as bispecific antibodies generated frorre at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies t the desired biological activity. An antibody can be of any the five major classes of globulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. lgGl, lgG2, IgGE, IgG4, lgAl and IgAZ), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known t structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

A "blocking" antibody or an ”antagonist" antibody is one which inhibits or reduces biological activity of the n it binds, such as CD37. in some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. The biological activity can be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

The term "anti-CD37 dy" or "an antibody that binds to CD3 7" refers to an antibody that is capable of binding CD37 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD37. The extent of binding of an anti-CD37 dy to an unrelated, non- CD37 protein can be less than about 10% of the binding of the antibody to CD37 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an dy that binds to CD37 has a dissociation constant (Kd) of :1 nM, £100 nM, 510 nM, 51 nM, or 30.1 nM.

W0 2012/135740 _ 14 _ The term "antibody fragment" refers to a portion of an intact antibody and refers to the nic determining variable regions of an intact antibody. es of antibody nts include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and pecific antibodies formed from antibody fragments.

A "monoclonal antibody" refers to a neous dy tion involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full—length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, inant expression, and transgenic animals.

The term "humanized antibody" refers to forms of non—human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that n minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are ed by residues from the CDR of a man species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FR) es of a human immunoglobulin are replaced with the corresponding residues in an antibody from a man species that has the desired specificity, affinity, and capability. The humanized dy can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or lity. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR s that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. es of s used to generate humanized antibodies are bed in US. Pat. 5,225,539. {0060} A "variabie region" oft-1n antibody refers tn the variable regien of the antibody Eight. chain or the variable region of the antibody heavy chain, either aicne er in combination. The variable regions of the heavy and Eight chain each consist at" four ork regions (FR) cnnnected by three complementarity determining regions {CDRs} also knnwn as hypervariabie regicns. The CDRs in each WO 35740 _ 15 _ chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contrébute to the formation of the antigen—binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross—species sequence ility (i.e., Kabat et al.

Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et a1 (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National utes of Health, da, Md. (1991). Using this numbering system, the actual linear amino acid sequence can n fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a ing to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of gy of the sequence of the antibody with a "standard" Kabat numbered sequence. Chothia refers d to the location of the ural loops (Chothia and Lesk J. Mol. Biol. 1962901-917 (1987)). The end of the Chothia CDR—HI loop when ed using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is e the Kabat numbering scheme places the insertions at H35A and H3 5B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and a ural loops, and are used by Oxford Molecular's AbM antibody modeling software.

W0 2012/135740 PCT/U82012/031648 _ 16 _ Loop Kabat AbM Chothia LE L24FL34 L24—L34 L24—L34 LE. 1450-1456 LEO-L56 L50—L56 L3 L89—L97 L89—L97 L89—L97 H1 BBL-H358 H26—H3SB H26—H32..34 (Kabat Numbering) H1 1131-1135 1126—1135 2 (Chothia Numbering) H2 5 1150—1158 HS2-1156 H3 1102 1132 1195—11102 The term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or ength antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

The term "chimeric antibodies" refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the le region of antibodies d from one species of mammals (e.g. mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody.

When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids osed by tertiary folding of a protein. Epitopes foréned from contiguous amino acids are typically retained upon n denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more y, at least 5 or 8-10 amino acids in a unique l conformation.

"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) arzd its binding partner (e.g., an antigen).

Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding y which s a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the iation nt (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity dies lly bind antigen slowly and tend to dissociate readily, whereas high—affinity antibodies W0 2012/135740 PCT/U82012/031648 -17_ generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding y are known in the art, any of which can be used for purposes of the present invention. Specific illustrative ments are described in the following.

"Or better" when used herein to refer to g affinity refers to a stronger binding between a le and its binding partner. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an y for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 HM.

By ”specifically binds," it is generally meant that an antibody binds to an epitope via its n binding domain, and that the bindirgg entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that e, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D." By "preferentially binds," it is meant that the antibody specifically binds to an e more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may react with the related epitope.

An antibody is said to "competitively inhibit" binding of a nce dy to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit g of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

The phrase "substantially similar," or "substantially the same", as used , denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an dy of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.

PCT/U82012/031648 _ 18 _ A polypeptide, antibody, polynucleotide, vector, cell, or composition which is ted" is a polypeptide, antibody, polynucleotide, , cell, or composition which is in a form not found in nature.

Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The term "immunoconjugate" or gate" as used herein refers to a compound or a derivative thereof that is linked to a cell binding agent (i.e., an anti—CD37 antibody or fragment thereof) and is defined by a generic formula: C—L-A, wherein C — cytotoxin, L = linker, and A = cell binding agent or anti-CD37 antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.

A "linker" is any chemical moiety that is capable of linking a compound, usually a drug, such thereof in a stable, as a sinoid, to a cell-binding agent such as an anti CD37 antibody or a fragment covalent manner. s can be susceptible to or be ntially resistant to acid—induced cleavage, light—induced cleavage, peptidase-induced cleavage, se-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hilic forms thereof as described herein and know in the art.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer e, but are not limited to, carcinoma, lymphoma, blastoma, a, and leukemia. "Tumor" and "neoplasm" refer to one or more cells that result from excessive cell growth or proliferation, either benign (noncancerous) or ant (cancerous) including pre-cancerous lesions. Examples of r" or "tumorigenic" diseases which can be treated and/or prevented include B-cell lymphomas including NHL, B-cell neoplasms, such as B-cell c precursor B—cell lymphoblastic leukemia/lymphoma and mature lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low— grade, intermediate—grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell ma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's ma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, and anaplastic cell lymphoma . Non-cancerous cells are cells that do not result in the , l9 - formation of tumors or neoplasms or the pment of . However, non—cancerous cells can contribute to disease, e.g., mune es, and include, for example auto-reactive B-cells.

The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term “tumor cell” will be modified by the term "non-tumorigenic" when referring solely to those tumor cells g the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

The term "autoreactive" refers to a cell, tissue, protein, antibody or other substance that produces an immune response directed against an organism's own cells, tissues, proteins, dies, or other substances.

The term "subject" refers to any animal (e.g., a mammal), ing, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.

Typically, the terms "subject" and "patien " are used hangeably herein in reference to a human Administration "in combination with" one or more farther therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional ents which are unacceptably toxic to a t to which the tbrrnulation would be administered.

The formulation can be sterile. {0082] An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be ined empirically and in a routine manner, in relation to the stated purpose. “he term "therapeutically effective amount" refers to an amount of an dy or other drug effective to "trea " a disease or disorder in a subject or mammal. In some embodiments, the therapeutically effective amount of the drug can reduce the number of B-cells; reduce the number of autoreactive B—cells; decrease the symptoms of disease; or slow the progression of disease. See the definition herein of "treating". A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective .

The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label can be W0 2012/135740 PCT/U82012/031648 - 30 _ detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an tic label, can catalyze chemical alteration of a substrate compound or composition which is detectable.

Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or er. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. Prophylactic or preventative measures refer to therapeutic measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of lactic or preventative measures include those prone to have the disorder and those in whom the disorder is to be prevented. In n embodiments, a subject is successfully "treated" if the patient shows one or more of the following: decreased B-cells; sed autoreactive B—cells; decreased B-cell activity; decreased aberzant B-cell activity; decreased non-malignant B—cells, decreased non-cancerous B- cells, reduced immunoglobulin level; reduced ity and ity; ement in quality of life; or some combination of effects.

"Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to rs of nucleotides of any length, and e DNA and RNA. The tides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If t, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide ents. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl onates, phosphotriesters, oamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal es, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forsns of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily t in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional tides, or can he conjugated to solid supports. The 5‘ and 3’ terminal 0H can be phosphorylated or substituted with amines or organic capping group moieties of from l to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynnoleotides can also n analogous forms ofribose or deoxyrihose sugars that are generally known in the art, including, for example, niethyl—, Ziflnallyl, W0 2012/135740 PCT/U82012/031648 _ 21 _ 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeiic sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("foranacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether ) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

The term "vector" means a construct, which is capable of delivering, and ally expressing, one or more gene(s) or ce(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to rs of amino acids of any length. The polymer can be linear or ed, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, ation, phosphorylation, or any other manipulation or modification, such as conjugation with a ng ent. Also ed within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the ptides can occur as single chains or associated chains.

The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more ces or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. s algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non- ng example of a sequence alignment algorithm is the algorithm described in Karlin et al, 1990, Proc. Natl. Acad. Sci, 4-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci, 90:5873- 5877, and orated into the NBLAST and XBLAST ms (Altschul et al., 1991, c Acids Res, 25:33 89-3402). In certain ments, Gapped BLAST can be used as described in Altschul et al., W0 2012/135740 PCT/U82012/031648 _ 22 _ 1997, c Acids Res. L25:3389~3402. BLAST—2, ASTLZL {Altschui et al., 1996, Memoirs in Brigg/raraiogv, 266:460-430), ALIGN, ALIGNQ (Genentech, South San Francisco, California) or Megalign {:DNAS’DXR) are additional publiciy available software programs that can be used to align sequences, In certain embodiments, the percent identity between two nucleotide ces is determined using the GAP matrix and a gap weight of 40, 50, 60, 70, or program in GCG software (e.g., using a dnaCMP 90 and a length weight of l, 2, 3, 4, 5, or 6). In n alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. M0]. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of l, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with e table, a gap length penalty of 12 and a gap penalty of 4.

Appropriate parameters for l alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment re are used.

In n embodiments, the percentage identity “X” of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the ent of the first and second sequences (as aligned by visual tion or a particular of residues in the second sequence. If the length sequence alignment program) and Z is the total number of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent ty of the second sequence to the first ce.

As a non-limiting example, whether any particular polynucleotide has a certain percentage least 90% identical, and in some sequence identity (e.g., is at least 80% identical, at least 85% identical, at embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, n, WI 53711).

Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to ine whether a particular ce is, for instance, 95% identical to a nce sequence ing to the present invention, the parameters are set such that the percentage of identity is ated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when W0 2012/135740 PCT/U82012/031648 _ 23 _ compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Identity can exist over a region of the ces that is at least about 10, about , about 40-60 residues in length or any irategral value therebetween, and can be over a longer region than 60-80 residues, for example, at least about 90-100 residues, and in some ments, the ces are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.

A "conservative amino acid substitution" is one in which one amino acid residue is ed with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, irecluding basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), ged polar side chains (e.g., glycine, asparagine, ine, , threonine, ne, cysteine), nonpolar side chains (e.g., alanine, , leucirge, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., ine, phenylalanine, tryptophan, histidine). For e, tution of a phenylalanine for a tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the ptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the CD37 to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well— known in the art (see, e.g., Brummell et al., m. 32: 1180-1 187 (1993); Kobayashi et al. Protein Eng. 12(10):879—884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

It is understood that wherever embodiments are described herein with the language “comprising,” otherwise ous embodiments bed in terms of “consisting of" and/or “consisting essentially of’ are also provided.

The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). 11. CD37 binding agents The present invention provides agents that specifically bind CD37. These agents are referred to herein as "CD37 binding agents." Exemplary CD37-binding agents have been described in US.

Published Application No. 2011/0256153, which is herein incorporated by reference in its entirety.

The full-length amino acid sequences for human, macaca, and murine CD37 are known in the art and also provided herein as represented by SEQ ID NOszl—3, respectively.

W0 2012/135740 PCT/U82012/031648 Human CD3 7: MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVL AISGIFTMGIALLGCVGALKELRCLLGLYFGMELLLFATQITLGILISTQRAQLERSLRDVVEKII'IQ KYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTI LDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELG FMTLSIFLCRNLDHVYNRLAYR (SEQ ID N031) Macaca mulatta CD37: MSAQESCLSLIKYFLFVFNLFFFVILGSLIFCFGIWILIDKTSFVSFVGLAFVi’LQIWSKV LAISGVFTMGLALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLQDIVEKTI QRYHTNPEETAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDS TILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLEL GFMTLSIFLCRNLDHVYNRLRYR (SEQ ID NO:2) Murine CD37 (NP_031671): MSAQESCLSLIKYFLFVFNLFFFVLGGLIFCFGTWILIDKTSFVSFVGLSFVPLQTWSKV LAVSGVLTMALALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLR TIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPFVPCSCYNSTATN DSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNIISIVGICLGVGL LELGFMTLSIFLCRNLDHVYDRLARYR (SEQ ID N023) In certain embodiments, the CD37 binding agents are antibodies, immunoconjugates or polypeptides. In some embodiments, the CD37 binding agents are humanized antibodies.

In certain embodiments, the CD37-binding agents are capable of inducing complement dependent cytotoxicity, Examples of CD37-binding agents that are capable of inducing complement ent cytotoxicy are sed, for example, in US. Published Application No. 2011/0256153, which is herein incorporated by reference in its entirety. For example, treatment of cells with the inding agents can result in CDC activity that reduces cell viability to less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40% or less than about 35% of the cell viability of untreated cells. Treatment of cells with the CD37—binding agents can also result in CDC activity that reduces cell viability to about 70-80%, about , about 50—60%, about 40—50%, or about 30-40% of the cell Viability of untreated cells. In some ular embodiments, the CD37-binding agents are capable of inducing complement dependent cytotoxicity in Ramos cells.

In n embodiments, the CD3 7-binding agents are capable of inducing antibody dependent cell mediated cytotoxicity . Examples of CD-37 binding agents that are capable of inducing antibody ent cell mediated xicity (ADCC) are disclosed, for example, in US. Published Application No. 2011/0256153, which is herein orated by reference in its entirety. For example, treatment of cells with the CD37-binding agents can result in ADCC ty that produces at least about W0 2012/135740 PCT/U82012/031648 .. 25 - %, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 60% cell lysis. Treatment of cells with the CD37- binding agents can result in ADCC activity that produces about 10-20%, about 20—30%, about , or about 40-50% cell lysis. ent of cells with the CD37-binding agents can also result in ADCC activity that produces about , about 20-50%, about 30-50%, or about 40-50% cell lysis. In some particular embodiments, the CD37-binding agents are capable of inducing ADCC in Daudi, Ramos, and/or Granata—S 19 cells.

In some embodiments, the CD37—binding agents are capable of inducing apoptosis. In some embodiment, the inding agents are capable of ng apoptosis in the absence of cross-linking agents. Examples of CD37—binding agents that are capable of inducing apoptosis in vitro in the absence of a cross—linking agent are disclosed, for example, in US. Published Application No. 2011/0256153, which is herein incorporated by reference in its entirety. For example, treatment of cells with the CD37- binding agents can induce apoptosis in at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% of cells. In some particular embodiments, the CD37-binding agents are capable of inducing sis in Ramos cells and/or Raji cells.

In some embodiments, the CD37-binding agents are e of depleting B—cells. In some embodiments, the B-cells are autoreactive B-cells. In some embodiments, the B—cells are not cancer cells.

In some embodiments, the B—cells are not turner cells. In same embodiments, the B-cells are not cancerous cells, In some embodiments, the B—cells overexpress C1337. In some embodiments, the B— cells do not overexpress CD37.

Treatment of cells with CD37-binding agents can result in ion of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or least about 75% of B—cells.

In some embodiments, the CD37-binding agents do not deplete T—cells under the same conditions in which B—cells are depleted. For example, treatment of cells with CD37—binding agents can result in depletion of less than about 20%, less than about 15%, less than about 10%, or less than about % of T-cells. In certain ments, the CD37-binding agents e at least about 25% of B—cells and deplete less than about 10% of T-cells. In certain embodiments, the CD37—binding agents e at least about 30% of B—cells and deplete less than about 5% of T—cells.

In some embodiments, the CD37-binding agents do not e monocytes under the same conditions in which B-cells are depleted. For example, treatment of cells with CD37-binding agents can result in depletion of less than about 20%, less than about 15%, less than about 10%, or less than about % of monocytes. In certain embodiments, the inding agents deplete at least about 25% of B-cells W0 2012/135740 _ 26 _ and deplete less than about 10% of monocytes. In certain embodiments, the CD37—binding agents deplete at least about 30% of B-cells and deplete less than about 5% of monocytes.

] In certain embodiments, conjugates or other agents that specifically bind human CD37 trigger cell death via a cytotoxic agent. For example, in certain embodiments, an antibody to human CD37 is conjugated to a maytansinoid that is activated in cells expressing the CD37 by protein internalization. In certain alternative embodiments, the agent or antibody is not ated to a maytansinoid or other cytotoxic molecule.

The CD37-binding agents include CD37 antibodies such as CD37-3, CD37—12, CD37-38, CD37-50, CD37-51, CD37—56 and CD37-57 and nts, variants and derivatives thereof. The CD37- g agents also include CD37-binding agents that specifically bind to the same CD37 epitope as an antibody selected from the group consisting of CD37-3, CD37—12, 8, CD37—50, CD37—51, CD37- 56 and CD37-57. The CD37-binding agents also include CD37-binding agents that competitively inhibit an antibody selected from the group consisting of CD37-3, CD37-l2, CD37-38, CD37-50, CD37-51, 6 and 7.

In some particular embodiments, CD3 7-binding agents can be characterized by their ability to bind chimeric CD37 ptides, including murine/human and macaca/human ic polypeptides desribed in US Published Application No. 2011/0256153, which is herein incorporated by reference in its entirety, and provided in the table below.

Chimeric Sequence EPoly- 3 MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV ;hCD37— M1 FTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRV QELVLRTIQSYRTNPDETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAH RVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGL QKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID 5 i; i NO. 184) imuCD37-NEISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNK R176 AQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPA SKAHIYREGCAQSLQ(sEQID No1851 A hCD37— 3MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV M45 LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRi 5DVVEKTIQKYGTNPEETAABESWDYVQFQLRCCGWHYPQDWQVLILRGNGSEAH ; RVPCSCYNLSATNDSTILDKVILPQLSRLGPRAKLRQTADICALPAKAHIYREGCAQS 1 LQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID 1M1qu1861 M 5 hCD37m MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV ECD- LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRV H45 QELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEP RVPCSCYNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICAVPAESHIYREGCAQG 1 LQKWHNNLI$1vGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID N0: = s 1871A AA hCD37m MSAQESCLSLIKYFLFVNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV ; ;‘ECD—H5 LAISGIFTVIGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERth “27.

W;—IQVELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEP RVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICAVPAESHIY’REGCAQG LQKWLHNNLISIVGICLGVGLLELG’FMTLSIFLCRNLDHVYNRLARYR (SEQ ID NO: ;188) ’ hCD37H“;MsAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV ECD-H4 LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRV s TIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPCE YNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICALPAKAHIYREGCAQS LQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID NO: ;189) 'iiémné‘i‘lw';MsAQESCLSLIKYFLFVFNLFFFVLWIWILIDKTSFVSFVGLAFVPLQIWSKV Mac4 LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLR: DVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAH RVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPAESHIYREGCAQGL QKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID NO: M3045 LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLR; DVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAH 5 '5RVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLj hCD37— ;MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV MacS FTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLR.

;DVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAH ; RVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPANSHIYREGCARSL; QKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID NO: ' 192) In some particular embodiments, the binding of the CD37-binding agents to CD37 does not require human CD37 amino acids 109-138. Thus, some CD37-binding agents bind to a polypeptide comprising the amino acid sequence of SEQ ID . In other embodiments, the binding of the CD37— binding agents to CD37 is disrupted by mutation of human CD37 amino acids 3. Thus, some CD37—binding agents do not bind to a polypeptide comprising the amino acid sequence of SEQ ID NO:185.

In some embodiments, the CD37-binding agents bind to a polypeptide of SEQ ID NO:184 and to a ptide of SEQ ID NO:186, but do not bind to a polypeptide of SEQ ID NO:185.

In some embodiments, the CD37—binding agents bind to a polypeptide of SEQ ID NO: I 87. In some embodiments, the CD37-binding agents bind to a polypeptide of SEQ ID NO:187 and a polypeptide of SEQ ID NO:l88. In some embodiments, the CD37—binding agents bind to a polypeptide of SEQ ID NO:187 and a ptide of SEQ ID NO:1 89.

In some ments, the CD37-binding agent binds to a polypeptide of SEQ ID NOzl90, but does not bind to a polypeptide of SEQ ID NOzl9l. In some embodiments, the CD37—binding agent binds to a polypeptide of SEQ ID N02192, but does not bind to a polypeptide of SEQ ID NOzl9l.

W0 20127135740 PCT/U82012/031648 _ 28 _ {1311119} CD37 peptide fragments to which certain {JEN-binding agents bind to e, but are not 11511 to, CD37 fragments ccmprising, ting essentiafiy Of, 01 consisting ot‘aminc acids 2004243 Qt" SEQ 173 NO: 5 amino acids 2132142213 or SEQ H3 NO:5, or amino acids 2216343 of SEQ 1‘13 N131. in some embot11nen1‘» the £133Lbinding agent is specfin(1111/ binds to :21 1311 C1337 epitQpe (50111151131111: amino acids 2029.43 of SEQ ‘13:? N13], In some embodiments, the binding, of the 13133 7—15111d1ng agent 10 CD37 requires 1115 acids 2132243 of SEQ ID ‘NO:1 1n scme embodiments, the binding 01': the (31337,- 15111111119, agen1 111 CD37 requires amine acids 2013—2213 0.1“ SEQ 1D 750:1. In some e1nbnd1n1en1s, the binding 15tts1e CD37~55111111ng event to (31133 7 requires 1111111115 acids 221-243 Qf SEQ H3 NO:1. 13} Exampies of CD37~b1ndit1gagents with the afctementioned b1n111ng prnperties are desctibed in US. Publiszmd Application No. 201 170256153, which1s herein incorpersted by reference111 11s ty. 113131211 The C1D37~b1nding agents also finciude (SEW—binding events that comprise the heavy and 119,111 chain CDR sequences of CD373, 131337—12, {1337,68 CD37—513,C1‘337«55 56 or {7133757, 71113111131157 and fight chain CDRS of CD3 7~3 8, {3337513, 151337—51, 13133766 and (33.1767 1501513111 related sequences T11e1efcre, the €1.33 7—151111111157, agents can 111515 comprise heavy and .11 ght chain CDR sequences 151211 cemprise a consensus sequence Obtained by ‘the alignment of CI.3'.37~58, (3.133760 C133751, CD37~ 56 and (71337—57. The CDR sequences of CD373, 7—12, (311337—38, 513 CD 75'1, {75337-56L1.) and 57 as weli as 15.11: sus sequence 01 U33733, CD3750 (£337—51, 131337-56 and C1337 57 ate (1153151151511 111 Tatfies 1 and 2 beiow.

Table 1: Variable heavy chain CDR amino acid sequences “ """ Anuboch E 5711611111 1 _11533... YHCDR3 C15373 new (sEQ 1‘15“ 1:1111‘1‘1‘15153511 {SEQ 1D GGYSLAH (“S‘EQ 11‘5‘‘N‘Q‘-6) CD37—12 E 1115mm {s1Q115 WINTWQESR61.15 115 13557171115 (15191511551159) N15.3.3 N5351‘). E .....

CD37—38 611115117115115‘1‘15 111576.1111115 1:11:15 1‘15““ 5 GYYGYGAWFVY (SEQ ID 3505.55.3. \Q5.53. ..... 515%“.-- CD37—50 5 51117111111 (SEQ 1D 161LYst11'1s11Q1‘1‘5‘“ GYYGYGAWFAY (SEQ 115‘ 1111135 \014:1 :NQ--55.)-.- CD3751 51555511711 (sEQ1D““51111?8115"1\1 5“1‘15GYYGFGAWFVY ~(‘S‘E‘Q‘1‘55““ N151.6) \Q1”) N018) CD3756W1(SEQ 1D GYYGFGAWFAY (‘S‘EQn5“ _m ).

CD37-57 3151‘Aw1-1(sEQ1D GYYGYGAWFAY (SEQ ID CONSENSUS sQEEA or (5111711 1 _ 61 1115? S15E15 151EGYYG[Y or FjGAWF[V or {8.1513113 NQ25);:115115V01” N} (SEQ 1.1.5 :AEY. (SEQ ID NO27) ##LNQ;2.65- ..... 5252-5 *SYGMS (SEQ ID ssQQsYWEPDWEQ .HSYYDTSVDY NQ571). 1.1 -_ ..-..#W1 _ 5--(_S__EQIDNQ173) 252-3 SYQMS (SEQ ID 1 1 HSYYDTSVDY (SEQ ID Table 2: Variable light chain CDR amino acid sequences Antlbod VLCDRI VLSD" "I:YLCDR3 CD37-3 ASINIRSNIA “SEQ YATNLAD ISILQ III") QHYWGTTWT (SEQID AIDNUL“ EN:O30)A A NQ:2__9__)__ 77777777 CD37-12 RASQ‘SVSTSSYSYEY3 S (SIQ II) QHSWEIPYT (SEQID NQ:”A.»AAAIAAAAAA} A ___________________________________________(SEQID AAAA__WEN033) 38 5 II") .I ASSSVTYIVIII (SEQ , E QQWISNPPT(SEQID I II) NQ:AA3A/1IA)AA 0:35) ENQ:AA3__6__)AAAAA _ "" ,, ICD37-50SSM SATSS‘VTY\«INISLQNTKIPY ISFQ II)“"""""E QQWSDNPPT (SEQ IDA 1 HINDI“ 14% 2‘039),, ized ;DISNIPY ISEQID "333.1322 ,,,,,, WWI __I__w ‘ CD37-51 SA’I‘SSVI‘YVIHISEQDISKLASISEQID IDN04III ND4") ":EQQWSSNPPT (SEQ IDN043_)_MI iCD37—56 I’SASSSVYMHISTQ SI). QQWISDPPT(SEQID ; Humanwr‘d DTSNLAS (SEQ II") N047) mew _MM 77777 CD37—57 SAISSVTY‘EIII ISEQ DISNLAS 3 . EQQWSDNPPT (SEQ ID I EDNDIIEILIMI ND49).. MIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII, Iiumanized DT‘SNL"\S ISEQ ID CONSENSUS SA["T""o"; S]SSVTYMHNTS=K Qr NELIii0} 1le QQW[1 or S][S or D][N or (SEQIDNAQ:52} III AYI(SEQ IQN0: 513 DPPT (SEQ AIADAANQ:_54I 252-3 RASQDISNYLN (SEQ;Y1%:me Ing If)? QQGNALPWT(SEQID IDNQ:III1I7II4)_MNDWINO 176) The CD37 binding molecules can be antibodies or antigen binding fragments that specifically bind to CD37 that comprise the CDRs of CD37-3, CD37-12, CD37—50, CD37—51, CD37—56, or CD37—57 with up to four (i.e., 0, l, 2, 3, or 4) conservative amino acid substitutions per CDR.

The CD37 binding molecules can se one of the dual variable light chains or variable heavy chains described herein. Antibodies and polypeptides can also comprise both a variable light chain and a variable heavy chain. The variable light chain and variable heavy chain sequences of murine, ic, and humanized CD37-3, CD37-12, CD37-50, CD37-51, CD37-56, and CD37-57 antibodies are provided in Tables 3 and 4 below.

TabIe 3: VariabIe heavy chain amino acid sequences Antlbody WAmmo SQ ID NO) muCD37-3 !KESGPGINAPSQSLSI‘CI ‘v SLIP SIIII I SIIVS‘WVRQI’PGKGLEWLGVIW wUDUSAIANYHMSIKKDIISIISQVILIxLNSIAQIAAIIIIIAAA'AIYYCAISGIAsYSIIIAIAAAAAA WE 11WGQG 8.8I888A{S10113N13888 E E QVQVNLNAAAYAPSQS1 SITLT‘VSG1'8'1II11‘8GV‘8‘8‘8’\71813P?18K8818‘88; .

E GDGSTNY1-1SALKSRL8118.881AHQKSQ‘VFLKLVNUQ11313.1‘1581‘88CAKGGY’8.8.A E111181101511VTV11183111) .88.) NQ.5111 -1 huCD3W '11 E GGG88N8I88888888888818.88888828SQVFLKLN88..LAAG8‘A8‘8-Y.CANGGY‘88...AQVQVQESGWN7V’APSQTLS11CTVNGFSL188GV888I8I8Q8>8>818888888888G8I'8'18I'.‘ E 8888GQG888/88/88{88G113N1388 II) huCD373v1 .1 EQIvG8/Q838GPGL8IAP8Q8 I S1‘1CTVSGFSL1TSG‘VSY‘JVRQPPGMN1:888(88888..

E C8DGSTNYHSSLKSRLSEK.88.88888808888888NSLTAADI8‘A‘I8‘88I8:AKGGYSE.A E 11‘8‘8'11131111V8888 {SEQ113NQ588 muCD3712 E QRYLV'Q‘GPELKKE‘GE8828888.SGYTFTKYKAG888W‘s/111211018181818<8888G 88I888NI8IG8:88<.\8888888888A8881'88A8I8A8I8Q88N888E1331“A8Y’FC’GRGTv E 888.88888888888888288:8 N0.593 78161371732 1311YLVQSL11311KK1’181E I‘VKISCK88888888888888888NI8I8<QAQ888888KWMG 88I888NI8‘G8388.\8888888888A8888‘88A8I8‘A8ILQ88NLN888:88A8YESGNGIE‘vI VAD'WUOGTTL1'V651‘81‘Q1.7.173 NQ 6888 ‘muCD37WD8IG8Q888G8>8>88888388288SLTCTVTGYS11SG1G‘8‘8IHW1RQ1PCIK181FW‘xEIYY 88.8SGG1D‘Y‘NPS1KSR1S11RDT8K‘NQFFLRL‘8‘8V188-888A8‘8Y1A81888IG8GE.

E__A8888I888GQG88.8I8-8Ag88Q188NG:11.11. .....

Q‘v’QLQ'ESCii‘1)1.\71\1‘S172SLSLTLTV'8'G'8I8.81S18118WHW1RQ1PGTNKLEWV111'8"" ILY’SGG11'3Y‘N1‘S1KSR1S1TRDTSK‘NQ111R1SSV1”113131,.581YY1-ARGYYGYG AWFY’YW '7 1"\ISA (SFQ 113N13: 13‘) E hu7CD37-38 Q888888.Y1.888883888888.8‘8I8G8 718118118HWERQTPA88888888888888 LYSGGFDY’NPSIRS181S1TR13TS18NQ1TLRLSS‘VIL\131A1YY1‘ARGYYGYG AWFY’YWGQG1L "7 ‘ 7711111513§Y1750 888Q8828881IW~ . 1YSGSTVYSPSLKSRlS11'RD1'SKNHFFLQLNSV1113131A1'YY’1‘A1118YY(1Y1: 1.A.WEAYYVGQGTLY’TVA (SEQ 1.1.3 N13643 . huCD3750 OYIQLOESAJ’GLLRRSQ‘AS1'1'CY7‘VSGYSITSG1AWHWERQHPGRKE..... .8 1.1.YSGST‘vY’SPSLKSRIS1”1RDTSKNHP1:1.Q1.NSVTAADTATYY”CARGY’YGYG ANY/1*AY’W1NY1zTLYTVSAgSEQ11318065) “““““““““““ huCD3751 .. IQ8.VFSGPEVLKPG1:88.81'88: “81">SG1"YYY11YY’1R01PGRGLE‘WRAG'Y}; HYSCxSTNYbPSLQGRISI11113881181314808N88I8A8838AI8888ARGYYGEGA 8I88IGQ88888888188A 13131:? 888 No67 EEEEEEE muCD3756 DVQLE‘ESGPDLVKPSOSES1.'8'GI1‘8'I"8G8I88I8‘8C8A88I888888Q8~1"(J\K18 88-88888 E (YAW‘8.A8EW880G888-11‘"8A888G11.3 N88 68)8888SQGTNY’TNPSLKSRVS11111.71.SNNQ811.QLNS‘VTTEDTATYYCARGYYGF - huCD3756 QVQLQESGFGLVK1’S13S1S1.”:11'1VS18Y’S1T‘8181'A‘818’11W1RQF"GKG11‘?88888888 HYSGGTNYNPSLKSRV81111111810811“:8‘8.Q8.NS‘JTAADTATYY 8::ARGY’YG8‘ GAVE-8A8 88888888888888A ‘88 111180 889) muCD3757E1‘1VQLQJ}.8G883888888888888'8..I88i'8I17I'1CY8888;.YFAWHWIRQE88888888:W7881888 E LYSGS.1VYSPSIKSR1S1TRD1'S1LNQ1‘1IL131.NSV11'1113TATYY1,I'YRGYYGYG 111 ‘ :- Y‘vijI‘xYWGOGNVFYSAESFQ .. 1'l75'7""""""""' QVQLQESGP1Y181.K1‘SQSISLTC1Y1S18Y S11"»G1IAW11‘8‘8’1RQF1318188:6771.FYY’MGYI' LYSGSFIS‘JY13881811SK1S11RDTS18NQFFLQLNSV1'AAD11A1.YY’CARGYY(1‘11: EVQ‘V 777712717. . .77 A81s1TFSSY1xY1S‘V8’Y’RQ11’11KR11YV’VA11 SSGGSYTYSPDSV181811.?11‘81813\AKK1'1.Y1.QMSSLKSEDTAMYYCARii-SYY’ _ 31 _ Table 4: le light chain amino acid sequences ““““:Q 1body“““‘“““““m“““““““““““““““““““““““““““““““““““““V1:1A1?)“11:11.1 {$.91} 11.“) N13) “V—‘EE“ muCD37-31D1QMIQS1’AS1EVSVAaFT‘YTE1‘L1111erN115:SNLAWYQQKQGKNPQLL‘YNYA1 1N1.ADGVFSRFSG‘YAYQIA1TQYSLKINSLQSE13F(11YYCQHYWAJTTWTEGGG115 1...... chCD°73 .. LIL:1571111.1LA511511:5151A5Y'QQKQLK5FQLLYNYA1 1'NLA1513YF5515551151.sTQ'Y5LK1N5LQ5F1::"1Fc11YYCQEWWGTTW111311511211 LUKRLSEQ 111110.73) Es huCD37—3 11115511115115 1..5Y<YL:1'KY'1'11L'KANLN . . ANYQQKFQK5FKL1YNYA1 (1.0 and 1.1) :NLADQYIFKKF'5L15Q5QYDY5LK1N5LQFF55FL1'1YYL'Q:1YYY11115711111151.:1'K LUKRWEQ117% 1 muCD37-12 :151Y11Q5FA51AY'5LL:QK-A115L'KANQ5 '5Y5YLYYY YEKFQQF T 1YA5N1A5<AIFAKF5LI56501151'1'LN1F1FY .;:1:~.F151A'1YY'CQF15WF1FY“1171.11.16 1 '1'KLL1KKE:5FQ 11.5%N15“75.5.? chcfi3“7“2““1“2““““““““““““““““§1.51571,1Q5FA5LAYSLLIQLLA1151KA5Q5Y'5'155Y5YLYYYYQQNFGQPPKU1K 51YASN1ASGYPAR¥5135135L11'1511'1.N111FV11'11YTAYYY'LQ115YYF1FY'1'FL1L1L‘1 -..:.-11\U'11\R 151531131???75>...- muCD37—38 “: Qi‘vi1Q5FA1NI5A5FIILKY'11511135A555YI'TY'N111YYYQQK5L1'1'5FKK5Y'1Y15'15 KLA5QY'FAKF5GGL:51:15Y 51:11SSMFA1111AA1Y’Y’CQQYY15N'FF'1'FL1<1Q'1'KL 8151515175} 017.11w 77777 chCD37—3 8 QW11QQ§PA1MSASPLYENV1N11L5A555Y'1YY111YY'YQQK511“1'5FKKYY1Y15“1'5 15LASGVFAK1\(YGC1‘5L1TSYSLIIS'SMAF1“)AATYYCQQWISNPF“FF(581.11K1 FEAR {<LQ115E5075) huCD37—38 “:“151571'1QYFA5515A5FGLKY'151“1“E“5A555Y1YN2111YYYQQKT’LYT<FKKYY1YQ1“5“" 1 :KL:A5L1YF.IAKF5L15<I5L1.155515155511;.AEDAA1Y7YLQQYY5NFF1'FLK1L11K1. 11?1.15.11 15LQ 115 NOE'EIjFE muCD37—50 1151531.“1QYFA1515A FL“ .KY1Y11L5A155171Y“511151:YQQK5LYT5FKKYAILY““1515““ 1K1.FYGYFL1111'5L15<I5L115Y5L11551113.IA515AA'1YYLQQYY515NFF1FG<1Q1KL 1.1:1KK15LQ 115 N0.5155 : huCD37—50 1'157L1QSPA11Y1‘5A‘5PG1KY515111:5.75.1555;1Y5I1HYYYQQKFQQYFKKYYLY155'“5““ 1:‘NLFYL1Y'FAKF5551.1551'SYSLTISSMEAEDAA1YYLQQ55511NFF1FGQL11'KL' KKY51'Q115 NQ51) 1 FAKF5115mm15Y'51.11<5MLIALLYIAA1'YYLQQYY55NFFTFQQQYKL. E .331.515(51:15 115 N1555E: _ EEE: muCD37—56“(11511511281311515A5FLI15KYTN11L5:A555Y1Y. . 'QQNZSGTSPKRWE‘Y’D’I’S i :KLASGVPARFSGGGSG1SYS11155;515:5115IAA'1'YY'L:QQW1515FF'1‘FL113GTKL <51'Q 11::N15 54) MM£115.15DIVLTQSPAFMSIANPLY‘FKV115111..5715555151MLNYYQQK1>15Q5FKKYY1Y1515EE huCD37—56 A5QYI'F5K1511QLY5Q1'15Y51115515111A515AA'1YYCQQW1515FF111:1st1KL L1KK :55Q- 115N133...“1 1.“5.’“51.3 ‘~“}1.“le Q15711QS1’A1‘Y1SASPL3EKY1511'L 5““1115557Y1V111AA’YQDKNG'1'5FAFYY1Y“151““5““ g KLASGYFAKF<1:51156155'5LT155Y1YIA515AA1YYLQQYA'5155FF1FL15L11K1: 1311513190115 N0256 huCD37_.57.::““ W0 2012/135740 PCT/U82012/031648 _ 32 _ ________________________________ PARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKL MINEIKR(SEQ ID NO:872 7777777 252—3 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTS i 3KLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPWTFGGGTKL ELISEQEQ112N9111§>m \\“\V_‘.“I....u.uu.........w......_______...r.r.\\ Also provided are polypeptides that comprise: (a) a ptide having at least about 90% sequence identity to SEQ ID NOsz55—7l or 177; and/or (b) a polypeptide having at least about 90% sequence identity to SEQ ID -87 or 178. In n embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% ce identity to SEQ ID NOS:55-87, 177, or 178. Thus, in cettain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NOsz55— 71 or 177, and/or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NOsz72-87 or 178. In certain embodiments, the ptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NOs:55-71 or 177; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NOs:72-87 or 178. In certain embodiments, the ptide is an dy and/or the polypeptide specifically binds CD37. In certain embodiments, the ptide is a murine, chimeric, or zed antibody that specifically binds CD37. In certain embodiments, the polypeptide having a certain percentage of sequence identity to SEQ ID NOss55—87, 177, or 178 differs from SEQ ID NOsz55-87 by conservative amino acid substitutions only.

Polypeptides can comprise one of the individual light chains or heavy chains described herein. dies and polypeptides can also comprise both a light chain and a heavy chain. The light chain and variable chain sequences of murine, ic, and humanized CD37—3, CD37—12, CD37-50, CD37-51, CD37-56, and CD3 7—57 antibodies are provided in Tables 5 and 6 below.

Table 5: Full-length heavy chain amino acid sequences “but.............,,."______ Ant1bodx \ FullLenvth Heav» Chain AmmoAcidSequence(SEQ IDNO) \ ,,muCD37—3 i QVQVKESGPGLVAPSQSLSITC‘VSGFSLTTSGVSWVRQPPGKGLEWLGVIW : GDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLA HWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTK VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVV . DVSEDDPDVQISWFVNNVEVHTAQTQTEREDYNSTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLT CMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN WVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ IDNO:88) § chCD37—3 QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIW GDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLA HWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS EWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT E KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV E WDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHL DVVTNETKEYKTTYVSNKATPAT’TTKTTSKAKT:QT’TETT’QVYTT.TTESRDETTKNQ T VSTTCT.VKCYTY’PSDTAVEVV’T‘S\TYQETNNYKTTT’T‘VLDSDQSTTLY’SKTTVDKT ‘ ‘TQKSTSTST’EIK YSTQ TD NQ:S9) T I"Hfiéfié'i'i'fiV‘TBT QVQV’. TTSLYVSVVVKQPEUKLYTEVVTUVTVV . VSGEST , . T GTTGSTNYTTPSLKSRLSTKKDTTSKSQV4TKT.NSTTT\ADTATYYCAKEEYYSEA HVVT.IQEYTTVTVSSIVSTKGESVTFEAT’SSKSTSGQTAATETCLVKDYiPEPVTVS \IVNSGALTSGVHTT‘T)AVEQSSLYLYSLSSVVTVT’SSST..(YT.Q.TYTCNVNTTKPSNT 3 KVETKKVEEKSCDKTHTEPECEAPELEGT‘YT’SVELEET’KT’KT)TT.TVTTSRTEEVTCVT V’TJVSTTEDT’LVKENVV’YVTTTJVTVTT’VAKTKPKFEQYNSTYRVV-SVLTVT.HQ T TNT/"LNGKEYKCKVSNKATPI‘TT’TEK'T‘TSKxKETQPRET’QVYTLT’PSRDELTKNQ VST.TCEVKGTYT’SETTAVEVVTLSNTJQEENNYKTTPEVTTTSDQSFEEYSKLTVBK SKVVQQTYNVESESVTYTTTT‘ALTTNTTY’TQKSLSTSEQ {SEQIT)NO99‘ at” huCD373V11 TQVQVQESGEGLVAPSQ”STTSTTETVSGTSLTTSGVSVVIRQT’EQKEYLEWTTIVTW g (TE)(.ESTNYT'TSSTKSKLSTKKDTTSKSQVE’T.KTNSTTAADTATYYCAKCTGYSLA HVVGQG’T‘LVTVSSASTKGPSVET’EAT’SSKS'T'SGG'T‘AAT..GCT.VKT)YEPEPV'T‘VS VTI'NSGAL'T'SC:V'TTTEE‘AVLQSSGLYSLSSVVTVESSSLCY'TQ'T‘YTCNVNTTK?SNT KV'DKKVF.T’KSCTBKTTT‘TCPPCPAPELEGGESVETEPT’KEKTT'T'EMTSRTETTVTCV VVDVSHEDEEVKENWYVDGVEVITTNAKTKEKEEQY’NS’T'YKVV’SV'LTVETITQ DVVLNGKEYKCKVSNKATEAE‘TEKTTSKAKGQT’RE.T’QVY"TT.T)T’SKTJETXTKNQ VSTTCLVKGEYT’STTTAVEWESNGQE‘FNNYKTTT’DVLDSETGSE‘ELYSKTXT‘VUK SRVVIQQGNVITSLSVVTTTEATTTTTHYTQKSTTSTST’G TST‘Q TD NQ19.1.). muCD3712 QTQLVQSTYPETKKETTE.TVKTSUYASLYYTTTKY’EyVTNVvVKQl—YQEIKTYTKW‘YTT: WTNTNTTJESKNAEEEKETKEAEST.ETSASTAYTQTNNEKYEDTATYEEGTEGTV VGQLYTTLTVSSAKTTAT’SVYET.AEVC{.YDTTGSSV’TEGCLVKGY’EPET-TV ’VVNSGSESSGVTTTET’AVEQSDTY'TLSSSV'T‘V‘TSSTVVI’T’SQSTTCNVATTT‘ASS TKVDKKTET’RGPTTKEE,T“PCKCPAE\LLUUESVETTT’T’KTKTYVTTVITTST.SPTVTCV VVDVSEDDT’TTVIQTSWEVINNV‘3VITTTAQTQTTTREDYNSTLKVVSATPTQHQT) VVJVITSTsKEEKCKVNNKDLT’AETERTTSKEKSV’RAPQVIYVLT’T’T’EEETVIT'T'KKQV TTTC.IVTVTDETVTEEDTYVEVV’TNNGKTET..NYKNTTPVTDSDQSYEMYSKLKVE KKNVYIVERNSYSCSVVTTETYLTDTTTTT’TT K (SEQ TDNQ92) __ __ YI12 ”I \ SGPETKKPGETVKTKEKASETY ’T Y T‘ "'YQMNTYIYKQAQQKQLKYYYKT WINTNT(YTSRNATEYKGKFA}SLEY8As.TAYY.QTNNTKYEDTIYTYECGRGTV V'ADVVI’GQG’T‘TTJT‘VSSASTKGPSVFPLAPSSKS’T‘SGG’T‘.IV.AT.GCT..-VKTTYFPTEPVT E SQALY‘KGYH‘T‘YPAVITQSSQLYSLSSWYYPSSSLQY'QYYTCNYNHKPS KKYEYKSCDKYH‘TCPPCYIAYELLQGYSVKLKPPKPKQYLMTSK'TYIKYI'Y" g CV’VVDVST-TEDPEVKT‘NWY’V’TXTVI‘EVHNAKTKPREEQY’NSTYRV’VSVL’T‘VL HQY‘YWTNQKEYKCKYSNKALKAYYKKYYSKAKQQYKEPQYYYLPYSKDELYK = NQVST.Y‘QLYK“TYPSDTAVI’TZWESNGQTTE‘NNYK‘T‘TPPVTDSTTGSFFLYSKLTV ' YYIQK (SEQ m NQ:93Y ‘ T KSTYVV’QQTYNYIESESV‘YTTTTZALTTNTTYT‘O I ‘ I: muCD3738 DVQTQFSGPDLVIKPSQSLSLTETVTTYYSTT QYYHYYIITRQFPGNKLEVV’MAY’ ; TYYSGETTDYNPSLKSRTSTTEDTSKNQFTI‘T.KY.SSVTTTLT)TATYY’CARGY’YGYG AYTIKVYIWGQQ‘T‘LYTYISAAKYYPPSYYYITAPGSAAQ'T'NSKITY'I'LQCLYKQY‘YY EEVI’T‘VI’T‘WNSGSTSS"G‘VI'TTTFEAV‘LESDLY’TLSSSV’T‘V’PSSMKPSE’T‘V‘TCNVAH 13ASSTKVDKKTVPRDCGCKPCTC’T‘V’PEVSSVETEPPKPKDV’LT‘T’T‘L’TT’KVI’CTCV‘V’ EVI’TJTSKTTDPEVQFSVVI’FV’DDV’E‘VI‘HTAQ'T‘QPRE.EQFNSTFRSVSELPTMHQDWL I NGKET’KTKYINSIYAFT‘APTEK'T‘TQK’T‘KGTTT’KVT’QV’Y'T'TPPPKTQMAKDKVSLT CMYTDFTPESTTV'EwQYYNQQYIAENYKNTQPTTVITNTNGSYTVIY’SKTNVQKSN V‘vEAEYSTFTESVETTETYLTTNTTTTTEKSTAHSETYK SEQ TD KY ' chCD3738 OVQLOESTJPDLVKESQSTST...TCTVTTYYSTTSEYF YTYIHWTKQ KLEWMAY T TE.YYQGTIYYSPYLKYKTYYYKDTSK\QEETKT.SSVTTEDTATY’YCARGY'YGYG AWEYIYIYVGQQ'IYYTVSAASTKGPSVT‘PLAPSSKS’T‘SGGTAATQC}V’KTJYEPE T PVTY’SVV’NSTYALTSGVHTEPAVTQSSGLYSTSSYYYYPSSSTQTQ“:YY’YCK-VNH KESNTKVT‘PI‘N:T j____1_g§g§§f_ .. APETTWETGPSVFTYYYKPKDYTMTYKYYT .....

VT.HQTJVV’LNGKEYKCTLVISNKYALEAT’TEKT1SKAKGQPKET’QV7Y1‘LET‘SRDHT3 TLNQVIST.1‘CT.YVKGE‘V‘PSDTAVEVVE‘SN GQE‘E‘I\‘YTV’KT1EE‘V71YDSDIGST‘TLVSKL1 V’DKSKWT‘QLYNV’E‘SCSV7V1TTEALTTNT1‘Y’1‘QKSLS1SEG (SEQT33NO:9;?) 3huCD3‘7l‘é‘53‘""""""""""1Q‘V"QT(3ESGE‘GTVKE‘SQSLSLTC1‘V"SC“:STTS(.TE{‘1‘V7171TVV’TRQT‘PGKYYLEVVI‘VLAV’ LVSGG1‘DYNEST.KSKTSTTRDTSKNQEELKLSSV71AAD1‘ATYYCY‘ARGV’V"GYG YAVVI‘EVI‘YVV’GQGTLV’‘T‘V/S SAS‘T‘KGT‘SV’EELAPSSKS‘T‘SGG‘T‘YAAT..G(‘.‘.LV’KT3YEPE E‘V‘TV’SVV’NSGAL‘T‘SGV‘TEET‘E‘T3AV’TYQSSC:LYSLSSV’V"TVI'T‘SS Q'T‘YTCNV‘NTT K1)SN'T‘KVI’TJTLKV’EPKSCDK‘T‘ETTCT‘T‘CPAE’E‘YTYTYGGPSVELET’PKEKDTTYMTSRT‘E’ V‘V’V1‘3V’STTTT3PEV7K1‘NVV’VVDCY‘V’EVHN‘YAKTKPKET‘Q‘V’NSTVKV‘V"3,71;T 7-LT1Q13WT.YNLYKEYKCKV’SNKALT‘AE’TEK1TSKYAKi.‘TQEKET—‘QV'YTEEPSKDEL1‘1 KNQVSLT‘C1V7K111“V’E‘S‘DTYAV’EVV’ESNLYQT‘E‘YV‘Y’ K1‘11’1’V1.1‘3S13GSTTTYYSKLT 111 111.1113 760 LEVI"QLQ “ ‘YYEDTTYKESQSLSLTCTV’TGI‘TSTT‘SGEYAVVITTVV’TRQT‘PLNKLEWMG‘YT LYSGST‘VI‘1’LT‘ST.KSRTSTTKDTSK\T11ELQLNSV’1‘1‘E1‘31‘YATY‘Y‘CARGYV’GYCY AWEYAYVVI‘GQG1‘T.YV’1‘V’ AE‘SVV’PLAPV’C.(1131‘TGSSVIT1.GCT.V’KGYT‘T‘ Y ET‘V’TLTVVNSUHSSGV’TT'.1‘EEAV’TQSDLV’TLSSSVITV’TSST‘VVIT‘SQSTTC‘NVATTT‘ 5; VI1C3KKTEE’KGT3T.1K1“CPE‘CKCPAT—‘NLLGGPS V7E‘TE‘T3T’K1K1‘3V’1.T\I11ISTYST—‘1V7 ‘ V7DV"SEDDPDV’QTSVV’EV’NNVEV’ETTYAQTQTT'TKEDV’NS‘T‘LK.VV"SYA1.YETQH QT3VV’TV’TSGKEFKCKVI'NNKJJLEAT‘TEKTTSKE‘KGSV‘RAPQV’V‘V’LET‘T‘EEEMTKK QYI1'11CVT'V‘TDFIVYPEDIYVEVV’TNAIGK’T‘FTY‘NV’KNTEE‘VI‘TDSDGSYT‘ME’SKEK \V’EKKTSVVVI’YKNYYSCSY’Y11C"'C11N11111I‘KSFSNTYCK5YYQ1QNQ;9. huCD3750 QYIQ1QESGEGLLKT’SQSESL1'C1YISG‘Y‘8.11811?AVVITTWKQHYICYNKC1i;YYYKYY 1 11Y SCYS1'YIYSPSTK151115111RD1SKN’KYFTYQ1.NSVTAADT‘ATYYCYARCYYYGVCY' .AVVIE‘A‘C VVGQGT1.VTV7SAASTTLGPSVE13LAESSKSTSGGTAAT.GCTV’KDYEPET1 PV’TVSWNSGAL’T‘SGV’E1"1‘T‘T‘YAV"1YQSSGLYSLSSV‘V’’T‘VI’ESSSLG‘T‘QTYTCNV’NH 1 * KPSNTKVDKKVEPKSCDKTHTCPPCPAE‘T‘TYLCYTYPSVIELEEEKT’KDTTVTTSRTP EY’11'.Y 11CDPCYIK1NYYY Y’QCYYI’EYINYIAKTKPKCCQYNYCYRVIVSV1'11 YIL11QDYYI1NCYKCYKCKYSNKALPAETEK’118KAKCYOT‘KT‘PQV’YTLPFSRDLL1' 1 KNQYISL1C1.V’KGEVE8131AV’EWES\GQ1‘E\1NYK111>1>YI113S13CSE‘EL‘YSKLT _____1__YIQ1<<YKYYIQQQ\Y " NTTYTQKSLSLSP6113113 NO93) ' ' I. . muCD3751 DYQLCLYCYYQYY. .'' BYSTSSGE‘AVVIHVV’TRQT‘EL.YNKLEVV7VTGVT TTYSGSTNYSESLKSRTSTTR13SSK‘YQFTLQL1YSVMiTEDTA.1YYC.YARGYV’GFGA WE‘VYWGQGTLV/1V7 SAYAKTTAT‘SV’VET.Y’AT’VCCYDTTGSSVTLGCT.YV’KG‘: T‘T‘EE’ VI"1“1..'T‘VV”NSGSLSSGV’T—TTE‘T‘AV’TYQST‘3LV’ 'T‘LS SSV’TV7TS STVVI‘T’SQST'T‘C‘NVI’AETT‘AS STKV‘1‘3KKTET‘KGPTTTLT-‘CT-‘PCKCEAT’N 1.YT.YGGESV7ETEPT’KTKDV’LMTSLST’TV’'T‘C VI‘V‘ V’ 1.3 V’SET31‘31‘T3VQTSVV‘FV71‘Y1‘1VTV7EV’ETTAQT‘Q'T‘ETKEDYNSTTYKV‘‘V7SYALT’TQT'1Q‘L3 ‘ WMSGKEEKCK‘V’N‘N‘KDLPAPTEKTTSKT’KGSV"RAT’Q‘V’YV71..-E1‘EEEETVTTKTLQV7 t TLTC.TVIT‘V’1‘T3FJVT1‘ET3TYV’EVVTTNNGKTLLNYKN'T‘ET‘V’TT3ST3GS‘YTVITYSKLKV‘ w—‘......./ :“BEEBWN .vEVQEVI‘ESGE’EV71K‘PLYT‘SLSLTLTVSTTY‘STSSGEAVV71TVVITRT‘TT‘T-‘(YKLYTEVVI‘V’TG‘YT TTYSGSTNYSPSTQTxKTSTTRDSSTNQT‘TLQLNSV’T‘ASDT‘YA1“!YT‘.KARGYYYL1EC§11A VVTV’YVV‘GQGTLV’T‘V‘SAYAS1‘KGESVFT—‘LAT—‘SSKSTSGGTAATGCLVIKD‘YEPE? 3 VTV’SVVINSGATYTSGV111‘E1’AVI1.QSSGT.Y‘Y’STSSVV7TV/‘ESSSLGTQ1‘YTCNVNETK KV13KKV’TTEKSCDTL‘TTTTLT’E‘CE‘YAT‘ETLGLYE‘SVETYT‘T‘T‘KPKL‘ITLMTSK11‘1: VIT‘CV’V’V’DVISELE1‘3PTEV7KENW‘YV’DG‘VTV’TTNAK.1‘KEREEQ‘CNST‘YIRV’V’SVLT VT.TTQDVVILNCKEVKCKV’SNKATPAE‘TEKTTSKAKCTQPKEE’QV‘VTTPE‘SKDTT.T: KNQV/STTCT.VKQT‘YT‘SDTAV’EV‘Y’SKGQPE\\VKTTYPVLDSDGSTELYSKL‘1‘ {WMVVVVVVVVVVVVVVVVVVVVVVVVV TTT‘Y’SGGEN17NEST1xSKV’STTRDTSKNQT‘T‘LQLNSVT1ET‘31‘ATYV’CAKGYYGT‘ GAVVIEAVVA7GQ€TTLVIT‘V"SYAAK1‘TE‘T’SV'V’E’T”A131TSAAQTNSMV1‘LGCLV’KGYE‘ "SSV’TV'PSSVTKE‘SL‘YT‘V:CNVIIA ............................................ GC‘KPCKTVPEVSSV’TTTT’T’KPKDV’TTTTTTPKVTCV T . SSTKV’DKKTVT’KDC SELT‘TMTTQTNV 1 VVDTSKT)T)PEVQTSVVTVIDDV’EV’HTAQTQPRTTQTNSTTRSV LNGKETKCRVNSAAPRAPTEKTTSKTKGKT’KAPQVYTTPPPKEQMAKDKVST TCMTTDPPPEDTTVEVVQVVNGQPAENYKNTQPTMNTNGSV’EVYSKLNV’QKSN 7 AGNTETCSVI’LHTC1TTTNHHTPKSLSTTSPC1K(SEQTD NC):NT.1} ‘ EV’QLQESGPGTVKPSQSLSLTCTV’SGYSTTSC717EA‘sV’HVVTRQEPGKT:LEWMGYT HYSGGTNYNPSLKSRV STTREPTSKNQTELGLNSVTTAAT)T7.ATYYCARGYYGE GAVVPPAYWGQGTLVPVSAASTKGPSVPPLAPSSKST7SGC3TAAT.GC’TV7KDY7EP TEPV’.7VS‘WNSC5IALTSGVHTEPAVT.QSSGT1‘17SLSSVVTVPSSSLGTQT‘17T7CNV7N E i TTKPSNT7KVTEKKVEPKSCDKTHTC7.T’PCPAPET.7LCsCsPSVPLEPPKPKDTTMTSKT T IPEVTCVV7VDV'SHEDPEVKENWYV’T)GVEV7HNAKTKPREEQYNS"1YRVV7SV7LTTVLHQDVVLNGKEVKCKVSNKALPAPTLKTTSKAKGQPRTPQVYTLPPSRDETTKNQV’SLPCLVKG‘TYPSTHAVEVVESNGQPENNYKT7T7PPVLT‘ASDGSETLYSKLE muCD1757 LYSGSTVV'SPST.KSRTSTTRDTSKNQTTLQLNSV7TTEDTA7TV7YC:ARGYYGYG AWEA717VV’V:QGTLVTV SAAKTTAPSVYPLAPVCGT)TT7GSSVTLGCLV’KGYEP i EPVTLTVVNSCTSLSSGVHTEPAVLQSDLYTLSSSVTVTSSTWPSQSTTCNVATTPE E DKKTEPKGPTTKPCPPCKCPAPNLLGGT’SVPTEPPKTKTDVT.MTSLSPTV E TCVVV7T3V7SE17DDPE3VQTSVV’EVNN7VEVPTTAQTQTTTREDYNSTTRVV78.ATPTQTT? QT)VVMSGKLTKCKVNNKT)LPAFTERTTSKPKGSVRAPQVYVLPPT‘EEEMT7KK QVTLTC‘.MVTHTMPEDTVVEWTNNGK7T7PT \YKNTEPVTDSDGSYEMYSKT.R VEKKNVV'1/1waSC511;ITELJLTT\TTTT DNGT93) 77E§5ET3§7~5 I I 77777§7QVQT 11 1. ‘KCYLT‘VVVTUYT 7VYSPSTKSRTSTTRDTSKNQPTLGTNSV’TAADTATYYCAKGYYGVG VVGQGTT.V7T7V7SAASTKGPSVPLVT’SSKSTSGGTAAT..GCLV’KDYEPE? PVTVSVVNSGALTSGVTTTET)AV’TQSSGT‘1SLSSVVTVPSSST.G77TQTYTCNVNTT KPSNTKVDKKVEPKSCDKTTTTCT’PCPAPET.LCsGPSVELEPPKPKL)TLMTSR7TP; EV77TCV’VVDVSHEDPCV’KTNVVYVDGVEV’T—TNAKTKPRECQYNSTYRVV’SVLT; VLTTQDVV’LNGKEYKCKVSNKIALPAPTEKTTSKAKCTQPREPQVYTLPPSRDELT771 KNQVSLTCLVIKGEYPSL‘AAVTVVTS‘TGQPENNYKTTPPVLDST‘GSEETYSKLTT 777TPVQVVESGGDLVKPGC 1 KTSCAASGPTTSSYGMSVVVKQTPDKKTEVV777VIACT SSGGSV’TYSPDSVKGRETTSRT)NAKKTL‘1LQMSSLKSET)TAMY‘7CARTTSY 7‘71 DTSVT)YVVGQGTSVTVSSAKTTAT‘SV'V’P7T.1APVCGDTTGSSV'TLGCTAI’KGYPP EPVTL'T'VVNSGSLSSGVT—TT‘PPAV’LQSDLV’TLSSSVTVTSSTWPSQSTTCNVAHP ASSTKV'DKKTEPRGPTTKPCPPCKCPAPNT.1T1GGPSVETEPPKTKDV’LMTSLST‘TV7 TCVVV’DVSETJUETJVQTSVVEVNNVEVTTTAQTQTHREDYNSTLKVVSALT’TQTT QDVVTVTSGKEPKCKVNNKTJLPAPTERTTSKPKGSV7K1APQV’V7VLPPPEEEMTKK CMVTDPMPEDTYV’EVVTNNGKTELNYKNTET’V7T.T.)ST)GSV’EMYSKLK ukwwmwmmummmmszm«11011111017% Table 6: Full-length light chain amino acid sequences 77DTQV’T’T QSPASLSV’SVGET \I . 7NV’AT NLADGVPSKESCzSGSGTQYSLKTNSLQSETEMPG777TVYCC‘HYV1CSTTWTTIGGGTKT LETKRADAAPTVSTPPT‘SSEQLTSC1CTASVVICPLNNPYPKBTNVKWKTDGSTRQ NGVLNSV‘VTDC.PDSKDST7‘17STVTSS771TT7TT.1TKT}PYERTT\TS‘1TCEAT7HKTS7TSPTVKST1 FNRNEQTVPQ 111 N1": 10¢: chCD373 2012/031648 -36— 'NLADGVPSRFSGSGSGTQYSLKFNSLQSEDFGTYYCQHYWGTTWTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQE SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKE SFNRGEC (SEQ ID NOJ91 ; huCD373 DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPKLLVNVAT E110 and 1.1) NLADGVPSRESGSGSGTDYSLK1NSLQPEDFGTYYCQHYWGTTWTEGQGTK; 1 LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ; E SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKE SFNRGEC(SEQID NO 1071 = DIV, QSPASLAVSLGQRATISCRASQSVSTSSYSYLY *QQKPGQPPKLLIK 71,,,..,,,,,,,,,,,,,,,,,,,,, EYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHS 11PYTFGGG TKLEIKRADAAPTVS11PPSSEQLTSGGASVVCFLNNEYPKDINVKWKIDGSE EVKSFNRNEC1‘SEQ1DRQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPINO11181 'Efiéfii‘i‘i‘z'" DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIK YASNLASGVPARFSGSGSGTDETLNILD’VEEEDTATYYCQHSWEIPYTFGGG ETKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA ELQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC1SEQID 1 _1 _____ ‘ébfil‘fi" QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTS EKLASGVPARFSGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKL DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN GEVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF E ENRNEC 1SEQ ID NO 110) chCD‘é‘YEEWmW1QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTS_ KLASGVPARFSGGGSG1SYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLE EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYEREAKVQWKVDNALQSE GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS; 111111 WENRGEC(SEQ 1DN0111) huCD3738 EDIVLTQSPASMSASPGERVTMTCSASSSVTYMHWYQQKPGTSPKRWXDTS ; EKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLI EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSE GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSE FNRGEC (SEQ ID NO 1 12) fimuCD‘i‘ii‘Sfiwm"E"Q1VLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTS KLPYGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKL E EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSEE?QN GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF _E NRNEC (_SEQID NO:113_1_ 757)‘ EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKKWHDTS E NLPYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSE . VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSE FNRGEC1SEQID NO 1141 777777 muCD3751 EQIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTS ; 1 KLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSNPPTFGSGTKL 1 EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN E : GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF huCD37-5 1 1EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTS KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGQGTKL EIKRTVAAPSVFIFPPSDEQLEKSGTASVVCLLNNFYPREAKVQWKVDNALQSWE W0 2012/135740 2012/031648 _37_ EGNSQE‘WTEQBS‘Kfifififs'sTLTLsflfiYEWKfiKV§Kf§i§fifi65§§fifii§W4 FNRQEC_SEQ ID NO: 116.: 1 muCD37-56 QIVLTQSPAFMSASPGDKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTS E KLASGVPARFSGGGSGTSYSLTISTMEAEDAATYYCQQWISDPP‘EtGGGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN EGVLNSWTDQDSKBSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC (SEQ ID NO 117) Ehucfig‘fig""""“""""‘E“DIVLTQSPAFMSASPGEKVTMTCSASSSVTYMHWYQQKPDQSPKRWIYDTS NLASGVPSRFSGGGSGTDYSLTISSMEAEDAATYYCQQWISDPPTFGQGTKL; EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSE: GENSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS ‘FNRGEC @139 ID NO 1 18) muCD3757 QlVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTS E KLASGVPARFSGSGSGTSYSLTISSMLAEDAATYYCQQWSDNPPTFGSGTKL EIEKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF huCD3757 EIVLTQSPATMSASPGERVWSSVTYMHWYQQKPGQSPRRWIYDTS PARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKL; EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNPYPREAKVQWKVDNALQSE GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSE FNRGEC(SEQ ID NO:___1_2__(_)) EEEEEEEEEEEEEEEEE 252-3 EDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTS VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPWTFGGGTKL EELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ : NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC“(SEQIDNQ: 180) Also provided are polypeptides that comprise: (a) a polypeptide having at least about 90% sequence identity to SEQ ID NOs:88-104 or 179; and/or (b) a polypeptide having at least about 90% sequence identity to SEQ ID NOS:105-120 or 180. In n embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs:88-l20, 179, or 180. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NOs:88- 104 or 179, and/or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NOs:105-120 or 180. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid ce of SEQ ID NOs:88-104 or 179; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NOs:105-120 or 180. In certain embodiments, the polypeptide is an antibody and/or the ptide ically binds CD37. In certain ments, the polypeptide is a murine, chimeric, or humanized arétibody that specifically binds CD37. In certain embodiments, the polypeptide having a certain percentage of ce identity to SEQ ID NOs:88-120, 179, or 180 differs from SEQ ID NOs:88- 120, 179, or 180 by conservative amino acid substitutions only.

In certain ments, the CD37 antibody can be the antibody produced from a hybridoma selected from the group consisting of consisting of ATCC t Designation PTA—10664, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10665, deposited with the ATCC -38— on February 18, 2010, ATCC Deposit Deisgnation PTA—10666, deposited with the ATCC on February 18, ‘10, ATCC Deposit Designation PTA~10667 deposited with the ATCC on February 18, 2010, ATCC Deposit Designation traumas, deposited with the ATCC on February 18, 20W, ATCC Deposit Designation P'T.A~i0669, deposited with the ATCC on February 18, 2010, and ATCC Deposit Designation PTA»10670, deposited with the ATCC. on February 18, 2010 {American Type Culture tion (ATCC) at 10801 University Boulevard, Manassas, Virginia 20E iii). In n ments, the antibody comprises the VH—CDRS and the VI..~CDRS of the antibody produced from a bydridoma selected from the group consisting ofPTA~10665, P13840666, VIA-10667, I’ll/7140668, PTA~10669, and PTA-10670.

In certain ments, the CD37 antibody can comprise a light chain encoded by the recombinant plasmid DNA phuCD37-3LC (ATCC Deposit Designation PTA-10722, deposited with the ATCC on March 18, 2010). In certain embodiments, the CD37 antibody can comprise a heavy chain encoded by the recombinant plasmid DNA phuCD37—3HCV.1.0 (ATCC Deposit Designation PTA-10723, deposited with the ATCC on March 18, 2010). In certain ments, the CD37 antibody can comprise a light chain encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722) and a heavy chain d by the recombinant plasmid DNA phuCD37-3HCV.l.0 (PTA—10723). In certain embodiments, the CD37 antibody can comprise the VL—CDRs encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722) and the VH-CDRs encoded by the recombinant plasmid DNA phuCD37-3HCV.1.0 (PTA- 10723).

Monoclonal antibodies can be prepared using oma s, such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma , a mouse, r, or other appropriate host animal, is immunized as described above to elicit the production by cytes of antibodies that will specifically bind to an immunizing antigen. cytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable a cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or s fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinant DNA methods as described in US. Patent 4,816,567. The polynucleotides encoding a monoclonal antibody are ed from mature s or hybridoma cell, such as by RT-PCR using oligonucleotide s that specifically ~39— amplify the genes ng the heavy and light chains of the antibody, and their ce is ined using conventional procedures. The isolated polynucleotides ng the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not ise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or nts thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 3482552- 554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can r be d in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a munoglobulin polypeptide to te a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density nesis of the variable region can be used to optimize specificity, y, etc. of a onal antibody.

In some embodiments, the monoclonal antibody against the human CD37 is a humanized antibody. In certain embodiments, such antibodies are used eutically to reduce antigenicity and HAMA (human anti—mouse antibody) responses when administered to a human subject. Humanized antibodies can be produced using various techniques known in the art. In certain alternative embodiments, the antibody to CD37is a human antibody.

Human antibodies can be directly ed using various techniques known in the art.

Immortalized human B lymphocytes zed in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol, 147 (1)186— 95; and US. Patent 5,750,373). Also, the human antibody can be selected from a phage library, where that phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat.

Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat’l. Acad. Sci., 95:6157162, Hoogenboom and , 1991, J. Mol, Biol., 2272381, and Marks et al., 1991, J. Mol. Biol, 222:581). Techniques for the tion and use of antibody phage libraries are also described in US. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2007, J. Mol. Bio., 376:1182 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., 1:992, Bio/Technolngy 10:779»783, incorporated by reference in its entirety) are known in the art and can be employed to generate high att‘inity human antibodies.

Humanized antibodies can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the e of endogenous globulin tion. This approach is described in US.

Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies that specifically recognize a CD37.

Bispecific antibodies are antibodies that are capable of cally recognizing and binding at least two different epitopes. The different epitopes can either be within the same molecule (e.g. the same CD3 7) or and bind a on ent molecules such that both, for example, the antibodies can specifically ize CD37 as well as, for example, 1) an effector le on a leukocyte such as a T—cell receptor (e.g. CD3) below. or PC receptor (e.g. CD64, CD32, or CD16) or 2) a cytotoxic agent as described in detail Exemplary bispecific antibodies can bind to two different epitopes, at least one of which ates in a polypeptide of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular n. These antibodies possess an antigen- binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodies are common in the art (Millstein et al., 1983, Nature 305:537—539; Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655—3659; Shalaby et al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; and US. Patent 5,731,168). Antibodies with more than two valencies are also contemplated.

For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodiments the antibodies to CD37 are multispecific.

In certain embodiments are provided an antibody fragment to, for example, increase tissue ation. s techniques are known for the tion of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (for example Morimoto et al., 1993, l of Biochemical and Biophysical Methods 242107-117; Brennan et al., 1985, Science, 229281). In certain embodiments, antibody nts are produced recombinantly. Fab, Fv, and scFv dy fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such antibody fragments can also be isolated from the dy phage ies discussed above. The dy fragment can also be linear dies as described in US. Patent 5,641,870, for example, and can be monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

W0 35740 _ 41 _ According to the present invention, techniques can be adapted for the productior’: of single- chain antibodies specific to CD37 (see US. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab sion libraries (Huse, et al., Science 246:1275-1281 (1989)) to allow rapid and effective identification of monoclonal Fab fragments with the d specificity for CD37, or derivatives, fragments, analogs or homologs thereof. Antibody fragments can be produced by techniques in the art including, but not d to: (a) a F(ab')2 fragment produced by pepsin digestion of an dy molecule; (b) a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

It can further be ble, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody nt or by incorporating the e into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide sis).

Heteroconjugate dies are also within the scope of the t invention.

Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells (US Pat. No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide ge reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methylmercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that provides for the association of the antibody with the polypeptides of a human CD37. In this regard, the le region can comprise or be derived from any type of mammal that can be induced to mount a l response and generate immunoglobulins against the desired antigen. As such, the variable region of the modified antibodies can be, for example, of human, murine, non—human primate (e.g. cynomolgus monkeys, macaques, etc.) or lupine origin. In some ments both the variable and constant s of the modified immunoglobulins are human. In other embodiments the variable s of compatible antibodies (usually derived from a non-human source) the can be engineered or specifically tailored to improve the g properties or reduce immunogenicity of the molecule. In this respect, variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid ces.

In certain embodiments, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs can be d from an antibody of the same PCT/U82012/031648 _ 42 _ class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of ent class and ly from an antibody from a different species. It is not alway necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the n binding capacity of one variable domain to r. Rather, in some cases it is only necessary to transfer those residues that are necessary to maintain the ty of the n binding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and ,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, those d in the art will appreciate that the modified antibodies of this invention will comprise antibodies (e.g., full-length antibodies or immunoreactive nts thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise d so as to provide desired biochemical characteristics such as reduced serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered nt region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region compatible witl‘: this invention comprise additions, deletions or tutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain nt domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, modified constant s wherein one or more domains are partially or entirely deleted are contemplated. In some embodiments, the modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted constant region domain will be replaced by a short amino acid spacer (e.g. 10 residues) that provides some of the lar flexibility typically imparted by the absent constant region.

Besides their configuration, it is known in the art that the constant region es several effector ons. For example, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also ates the inflammatory se and can also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a PC receptor site on the antibody Fc regior: binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse ical responses including engulfment and destruction of dy-coated particles, clearance of immune complexes, lysis of antibody-coated target — 4C\l—J eeliis by kiiier cells (called antibody-dependent cell—mediated cytotoxicity, or ADCC), reiease of inflammatory mediators, placental transfer and control ofimrnunogiobuiin production.

{GM-’35} in certain embodiments, the CD37~binding dies provide for altered effector functions that, in turn, affect the. biologicai profiie of the. administered antibody. For example, the deietion or inactivation (through point mutations or other means) of a constant region domain can reduce Fe receptor binding of the circulating modified dy. in other cases, it. can be that constant region modifications, consistent with this invention, moderate compiement binding and thus reduce the serum half iife and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or dy flexibility. rly, modifications to the constant region in ance with this invention can easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled n. gotiiztti} In certain embodiments, a CD3 7—binding agent that is an antibody does not have one or more effector functions. For ce, in some embodiments, the antibody has no antibody—dependent cellular cytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind to an F0 receptor and/or complement factors. In n ments, the antibody has no effector function.

It will be noted that in certain embodiments, the modified antibodies can be ered to fuse the CH3 domain ly to the hinge region of the respective modified antibodies. In other consteucts it can be desirable to provide a peptide spacer between the hinge region and the d CH2 and/or CH3 domains. For example, compatible constructs could be expressed wleerein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5—20 amino acid spacer. Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.

However, it should be noted that amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain ments, any omitted altogether, so as to spacer added to the construct will be relatively non-immunogenic, or even maintain the d biochemical qualities of the modified antibodies.

Besides the deletion of whole constant region domains, it will be appreciated that the antibodies of the present invention can be provided by the partial deletion or substitution of a few or even a single amino acid. For example, the on of a single amino acid in selected areas of the CH2 domain can be enough to ntially reduce Fc binding. Similarly, it can be desirable to simply delete that part of one or more constant region domains that l the effector function (e.g. complement CLQ binding) to be modulated. Such partial deletions of the nt s can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the PCT/U82012/031648 , 44 _ subject constant region domain intact. er, as d to above, the constant regions of the disclosed antibodies can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and genic profile of the modified antibody. Certain embodiments can comprise the addition of characteristics such as decreasing or one or more amino acids to the nt region to enhance desirable increasing effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific sequences derived from selected nt region domains.

The present invention further embraces ts and equivalents which are substantially homologous to the chimeric, humanized and human antibodies, or antibody fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution acid of an amino acid with another within the same general class such as, for example, one acidic amino with another acidic amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by another neutral amino acid. What is intended by a vative amino acid tution is well known in the art.

The polypeptides of the present invention can be recombinant polypeptides, natural polypeptédes, or synthetic polypeptides comprising an antibody, or fragment thereof, t a human CD37. It will be recognized in the art that some amino acid sequences of the invention car; be varied without significant effect of the structure or function of the protein. Thus, the invention r includes variations of the polypeptides which show substantial activity or which include s of an antibody, or fragment f, against CD37 protein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.

The polypeptides and analogs can be r modified to n additional chemical es the ical half not normally part of the n. Those derivatized moieties can improve the solubility, life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of proteins and the like. An overview for those moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

The isolated polypeptides described herein can be produced by any suitable method known in the art. Such s range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those ces in a suitable transformed host.

In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild—type protein of interest. Optionally, the sequence can be W0 2012/135740 PCT/U82012/031648 mutagenized by site-specific mutagenesis to provide functional s thereof. See, e.g. Zoeller et al., Proc. Nat’l. Acad. Sci. USA 81:5662-5066 (1984) and US. Pat. No. 585.

In some ments a DNA sequence encoding a polypeptide of interest would be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the d polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to size an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back— translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small ucleotides coding for portions of the d polypeptide can be sized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary asembly.

Once assembled (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequences encoding a particular isolated polypeptide of interest will be inserted into an expression vector and operatively linked to an expression l ce appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active ptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies, or fragments thereof, against human CD37. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an D37 antibody, or fragment thereof, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect ts having genes. A transcriptional unit lly comprises an assembly of (1) a genetic element or a regulatory role in gene expression, for example, transcriptional promoters or ers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate ription and translation initiation and termination sequences, as described in detail below. Such regulatory elements can e an operator sequence to control ription. The ability to ate in a host, usually conferred by an origin of replication, and a ion gene to facilitate recognition of ormants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the ._ 45 a if it is positioned so as to sequence; or a ribosome binding site is operatively linked to a coding ce permit translation. Structural elements intended for use in yeast expression systems include a leader enabling extracellular secretion of translated protein by a host cell. sequence Alternatively, where recombinant protein is expressed t a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to e a final t.

The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors sing expression control sequences from SV40, bovine papilloma virus, irus and cytomegalovirus. Useful expression vectors fer bacterial hosts include known bacterial plasmids, such as plasmids from Esherichia coli, including pCR l, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA phages.

] Suitable host cells for expression of a CD37-binding polypeptide or antibody (or a CD37 n to use as an antigen) include prokaryotes, yeast, insect or higher eukaryotic cells under the l of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as bed below. Cell-free translation s could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are bed by Pouwels et al.

(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the nt disclosure of which is hereby incorporated by reference. Additional information regarding methods of protein production, including antibody production, can be found, e.g., in US. Patent Publication No. 2008/0187954, US. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated by reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageously employed to express inant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly , riately modified and completely onal.

Examples of suitable ian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression s can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

The proteins produced by a ormed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, ential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, r magnetic resonance and x-ray crystallography.

For example, supematants from s which secrete recombinant protein into e media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the tration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matréx or ate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, e, dextran, cellulose or other types commonly employed in protein purification.

Alternatively, a cation exchange step can be ed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl . y, one or more reversed-phase high performance liquid chromatography (RP—HPLC) steps employing hobic RP-HPLC media, be ed to further purify a CD37- e.g., silica gel having pendant methyl or other aliphatic groups, can g agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous inant protein. inant protein ed in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting—out, aqueous ion ge or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of a recombinant n can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteins also include, for example, those described in US. Patent Publication No. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein in its entirety.

In certain embodiments, the CD37—binding agent is a polypeptide that is not an antibody. A variety of methods for identifying and producing non-antibody polypeptides that bind with high affinity to a n target are known in the art. See, e.g., Skerra, Curr. Opin. hnol, 18:295-304 (2007), Hosse et al., Protein Science, 27 (2006), Gill et al., Curr. Opin. Biotechnol, 17:653-658 (2006), Nygren, FEBS J., 275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by W0 2012/135740 reference herein in its entirety. In certain embodiments, phage display techeology has been used to identify/produce the CD37-binding polypeptide. In certain embodiments, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In n embodiments, the agent is a small molecule. atorial chemistry libraries and techniques useful in the identification of non- protein CD37-binding agents are known to those skilled in the art. See, e.g., Kennedy et al., J. Comb.

Chem, 10:345—3 54 (2008), Dolle et al, J. Comb. Chem, 9:855—902 (2007), and Bhattacharyya, Curr. Med.

Chem., —404 (2001), each of which is incorporated by reference herein in its entirety. In n further embodiments, the agent is a carbohydrate, a aminoglycan, a glycoprotein, or a proteoglycan. in certain embodiments, the agent is a nucleic acid aptamer. Aptamers are polynucleotide molecules that have been selected (e.g., from random or mutagenized pools) on the basis of their y to bind to another molecule. In some embodiments, the r comprises a DNA polynucleotide. In certain alternative embodiments, the aptamer comprises an RNA polynucleotide. In n embodiments, the aptamer comprises one or more modified nucleic acid residues. Methods of generating and ing nucleic acid aptamers for binding to proteins are well known in the art. See, e.g., U.S. Patent No. ,270,163, U.S. Patent No. 5,683,867, U.S. Patent No. 5,763,595, U.S. Patent No. 6,344,321, U.S. Patent No. 7,368,236, U.S. Patent No. 5,582,981, U.S. Patent No. 5,756,291, US. Patent No. 5,840,867, U.S.

Patent No. 325, U.S. Patent No. 7,329,742, International Patent Publication No. WO 02/077262, International Patent Publication No. WO 03/070984, U.S. Patent ation Publication No. 2005/0239134, U.S. Patent Application Publication No. 2005/0124565, and U.S. Patent Application Publication No. 2008/022773 5, each of which is incorporated by reference herein in its entirety.

III. Immunoconjugates The present invention is also directed to conjugates (also referred to herein as immunoconjugates), comprising the anti-CD37 antibodies, antibody fragments, and their onal lents as disclosed herein, linked or conjugated to a drug or prodrug. Suitable drugs or prodrugs are known in the art. The drugs or prodrugs can be cytotoxic agents. The cytotoxic agent used in the cytotoxic conjugate of the present invention can be any compound that results in the death of a cell, or s cell death, or in some manner decreases cell viability, and includes, for example, maytansinoids and maytansinoid analogs. Other suitable cytotoxic agents are for example benzodiazepines, taxoids, CC- 1065 and 5 analogs, duocarmycins and mycin analogs, enediynes, such as calicheamicins, dolastatin and dolastatin analogs including auristatins, tomaymycin ties, leptomycin derévaties, methotrexate, cisplatin, carboplatin, daunorubicin, doxombicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholino doxorubicin.

W0 2012/135740 PCT/U82012/031648 _ 49 _ Such conjugates can be prepared by using a linking group in order to link a drug or prodrug to the antibody or onal equivalent. Suitable linking groups are well known in the art and include, for example, disulfide groups, thioether groups, acid labile , photolabile , peptidase labile groups and esterase labile groups.

The drug or prodrug can, for example, be linked to the anti-CD37 antibody or fragment thereof through a disulfide bond. The linker molecule or crosslinking agent comprises a reactive chemical thereof. The ve chemical groups for group that can react with the anti-CD37 antibody or fragment reaction with the cell-binding agent can be N-succinimidyl esters and N—sulfosuccinimidyl . onally the linker molecule comprises a ve chemical group, which can be a dithiopyridyl group that can react with the drug to form a disulfide bond. Linker molecules include, for example, N- succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al., m. J., 173: 723-737 (1978)), N—succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., US. Patent No. 4,563,304), N- succinimidyl 4-(2—pyridyldithio)2—sulfobutanoate (sulfo—SPDB) (see US Publication 'No. 20090274713) , N—succinimidyl 4-(2-pyridyldithio) oate (SPP) (see, e.g., CAS Registry number 341498-08—6), 2- iminothiolane, or acetylsuccinic anhydride. For example, the antibody or cell binding agent can be modified with crosslinking reagents and the antibody or cell binding agent containing free or protected thiol groups thus derived is then reacted with a disulfide- or thiol-containing maytansinoid to produce conjugates. The conjugates can be purified by chromatography, including but not limited to HPLC, size- exclusion, adsorption, ion exchange and affinity capture, dialysis or tial flow filtration.

In another aspect of the present invention, the anti-CD37 antibody is linked to cytotoxic drugs via disulfide bonds and a polyethylene glycol spacer in enhancing the potency, solubility or the efficacy of the immunoconjugate. Such cleavable hydrophilic linkers are described in W02009/0134976. The additional benefit of this linker design is the desired high monomer ratio and the minimal aggregation of the antibody-drug conjugate. Specifically contemplated in this aspect are conjugates of cell-binding agents and drugs linked via disulfide group (-S-S—) bearing polyethylene glycol s ((CH2CH20)H=H4) with a narrow range of drug load of 2-8 are bed that show relatively high potent biological activity toward cells and have the d biochemical properties of high conjugation yield and high monomer ratio with minimal protein ation.

Specifically contemplated in this aspect is an anti-CD37 antibody drug conjugate of formula (I) or a conjugate of formula (1'): CB—[X1+CH24:H2CHHNY—D]m (I) -CH2—CHZO~~~)n—X1]m-CB (1') wherein: CB represents an anti-CD37 dy or fragment; D represents a drug; X represents an aliphatic, an ic or a heterocyclic unit attached to the cell—binding agent via a thioether bond, an amide bond, at carbamate bond, or an ether bond; Y represents an aliphatic, an aromatic or a heterocyclic unit attached to the drug Via a disuifide bond; {39:75} i is =3 or t; {00176} m is an integer from 2 to 8; and {00177} n is an integer from 1 to 24. {00178} In some embodiments, m is an integer from 2 to 6. {00:79] In some embodiments, m is an r from 3 to 5. {00180} In some embodiments, n is an integer form 2 to 8. Aitematively, as disclosed in, for example, US. Patent No. 6,441,163 and 7,368,565, the drug can be first modified to introduce a reactive ester suitable to react with a cell-binding agent. Reaction of these drugs ning an activated linker moiety with a cell-binding agent es another method of producing a cell-binding agent drug conjugate.

Maytansinoids can also be linked to anti—CD37 antibody or nt using PEG linking groups, as set forth for example in US. Patent 6,716,821. These PEG non-cleavable linking groups are e both in water and in non-aqueous ts, and can be used to join one or more cytotoxic agents to a cell g agent. Exemplary PEG linking groups include heterobifunctional PEG linkers that react with cytotoxic agents and cell binding agents at opposite ends of the linkers through a functional sulthydryl or disulfide of the synthesis of a cytotoxic group at one end, and an active ester at the other end. As a general example conjugate using a PEG g group, re “erence is again made to US Patent 6,716,821 which is ineoiporated entirely by reference herein. Synthesis begins with the on of one or more cytotoxic agents hearing a reactive PEG moiety with a ceii—‘oinding agent, resulting in dispiacernent of the terminal active ester of each reactive PEG moiety by an amino acid e of the ceh binding agent, to yield a cytotoxic conjugate comprising one or more cytotoxic agents eovaientiy handed to a eeii binding agent through 21 PEG iinking group, ativciy, the cell binding can be modified with the hifiinctionai PEG crossiinker to introduce a reactive- disultide moiety (such as a pyridyidisttifide), which can “then he treated with a thioi—eontaining niaytansinoid to provide a conjugate. in another method, the ceii binding can he modified with the bit‘ttnctionai PEG crosstinker to introduce a thiol moiety which can then. can he treated with 2t reactive disuttide-containing tnaytansinoid (such as a idisuitide), to provide a conjugate.

Antibody-maytansinoid conjugates with non—cleavable links can also be prepared. Such crosslinkers are bed in the art (see US Publication No. 69933) and include but are not limited to, N—succinimidyl 4—{maleimidomethyl) cyclohexanecarboxylate (SMCC). In some embodiments, the antibody is d with crosslinking reagents such as succinimidyl 4-(N-maleimidomethyl)— cyclohexane—l—carboxylate (SMCC), sulfo-SMCC, maleimidobenzoyl—N-hydroxysuccinimide ester W0 35740 PCT/U82012/031648 _ 51 _ (MBS), sulfo-MBS or succinimidyl-iodoacetate, as described in the literature, to introduce l-lO ve Hashida et al, J. Applied Biochem., 56—63 groups (Yoshitake et al, Eur. J. m., 101 :395—399 (1979); (1984); and Liu et a1, Biochem., 18:690-697 (1979)). The modified antibody is then reacted with the thiol- containing maytansinoid derivative to produce a conjugate. The conjugate can be purified by gel filtration through a Sephadex G25 column or by dialysis or tangential flow filtration. The modified antibodies are treated with the thiol—containing maytansinoid (l to 2 molar equivalent/maleimido group) and antibody- sinoid conjugates are purified by gel filtration through a ex G—25 column, chromatography on a c hydroxyapatite column, dialysis or tangential flow filtration or a ation of methods thereof. Typically, an average of 1-10 maytansinoids per antibody are linked. One method is to modify antibodies with succinimidyl 4-(N—maleimidomethyl)—cyclohexanecarboxylate (SMCC) to introduce maleimido groups followed by on of the modified antibody with a thiol-containing maytansinoid to give a thioether—linked conjugate. Again conjugates with 1 to 10 drug molecules per antibody molecule result. Maytansinoid conjugates of antibodies, antibody fragments, and other proteins are made in the same way.

In another aspect of the invention, the CD37 antibody is linked to the drug via a non— cleavable bond through the intermediacy of a PEG spacer. Suitable crosslinking reagents comprising hydrophilic PEG chains that form linkers between a drug and the anti-CD37 dy or fragment are also well known in the art, or are commercially available (for example from Quanta ign, , Ohio), Suitable PEG-containing crosslinkers can also be synthesized from commercially available PEGs themselves using standard synthetic chemistry techniques known to one d in the art. The drugs can be reacted with bifunctional PEG-containing cross linkers to give compounds of the following formula, Z X1 ( CH2 CHz—O )n Yp D, by methods described in detail in US Patent Publication 74713 and in W02009/0134976, which can then react with the cell binding agent to provide a conjugate.

Alternatively, the cell binding can be modified with the tional PEG inker to introduce a thiol- reactive group (such as a maleimide or haloacetamide) which can then be treated with a thiol-containing maytansinoid to provide a conjugate. In another method, the cell binding can be d with the bifunctional PEG crosslinker to introduce a thiol moiety which can then be treated with a reactive maytansinoid (such as a maytansinoid bearing a maleimide or haloacetamide), to provide a conjugate.

Accordingly, another aspect of the present invention is an anti-CD37 antibody drug conjugate of formula (II) or of formula (E'): CB—[XH—CHz—CHZ--»O—),,—Yp—D]m (I?) [D-Yp—(—CH2—CH2—O—)n—X1]m—CB (if) wherein, CB represents an anti-CD37 antibody or nt; {00184} D represents a drug; .. 52 ..

X represents an aliphatic, an aromatic or a heterocyclic unit bonded to the cell—binding agent via a her bond, an amide bond, a carbamate bond, or an ether bond; ] Y represents an aliphatic, an aromatic, or a heterocyclic unit bonded to the drug via a covalent bond selected ferom the group consisting of a thioether bond, an amide bond, a carbamate bond, an ether bond, an amine bond, a carbon-carbon bond and a hydrazone bond; 1 is 0 or 1; p is 0 or 1; m is an integer from 2 to 15; and n is an it‘steger from 1 to 2000.

In some embodiments, m is an integer from 2 to 8; and In some ments, n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer from 2 to 8. Examples of suitable PEG-containing linkers include linkers having an N-succinimidyl ester or N—sulfosuccinimidyl ester moiety for reaction with the D37 antibody or fragment thereof, as well as a maleimido— or haloacetyl-based moiety for reaction with the compound. A PEG spacer can be incorporated into any crosslinker known in the art by the methods described herein.

Many of the s disclosed herein are bed in detail in US. Patent Publication Nos. 20050169933 and 74713, and in WO2009/0134976; the contents of which are entirely orated herein by nce.

The present invention es aspects wherein about 2 to about 8 drug molecules ("drug load"), for example, maytansinoid, are linked to an anti-CD37 antibody or fzagment thereof. "Drug load", as used herein, refers to the number of drug les (e.g., a maytansinoid) that can be attached to a cell binding agent (e.g., an anti-CD37 antibody or fragment thereof). In one aspect, the number of drug molecules that can be ed to a cell binding agent can average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). Nzi—deacetyl-Nzi—(3-mercapt0-l-0x0propyl)- maytansine (DM1) and NT-deacetyl-NT-(4-mercapto—4—methyloxopentyl) maytansine (DM4) can be used.

Tiéius, in one aspect, an immunocongugate comprises 1 maytansinoid per antibody. in another aspect, an immunocongugate comprises 2 maytansinoids per antibody. In another aspect, an immunocongugate comprises 3 maytansinoids per antibody. In another aspect, an immunocongugate comprises 4 maytansinoids per antibody. In another aspect. an immunocongugate comprises 5 _ 53 _ maytansinoids per antibody. In another aspect, an immunocongugate comprises 6 maytansinoids per antibody. In another aspect, an immunocongugate comprises 7 sinoids per antibody. In another aspect, an immunocongugate comprises 8 maytansinoids per dy.

In one aspect, an immunoconjugate comprises about 1 to about 8 maytansinoids per antibody.

In another aspect, an immunoconjugate comprises about 2 to about 7 maytansinoids per antibody. In another aspect, an immunoconjugate comprises about 2 to about 6 maytansinoids per dy. In another aspect, an immunoconjugate comprises about 2 to about 5 maytansinoids per antibody. In another aspect, an immunoconjugate comprises about 3 to about 5 maytansinoids per antibody. In another aspect, an immunoconjugate comprises about 3 to about 4 maytansinoids per antibody.

] In one aspect, a ition comprising immunoconjugates has an average of about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1) drug molecules (e.g., maytansinoids) attached per dy. In one aspect, a composition comprising immunoconjugates has an average of about 1 to about 8 drug molecules (e.g., maytansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an e of about 2 to about 7 drug molecules (e.g., maytansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 2 to about 6 drug molecules (e.g., maytansinoids) per antibody. In one , a composition comprising immunoconjugates has an average of about 2 to about 5 drug molecules (e.g., maytansinoids) per antibody. In one , a composition comprising conjugates has an average of about 3 to about 5 drug molecules (e.g., maytansinoids) per antibody. In one , a composition sing immunoconjugates has an average of about 3 to about 4 drug molecules (e.g., maytansinoids) per antibody. {00201} in one aspect, a composition comprising conjugates has an average of about 2 d: 0.5, about 3 :1: 0.5, about 4 i 0.5, about 5 i 0.5, about 6 I 0.5, about .7 ;+.- 0.5, or about 8 i 0.5 drug molecules (e.g., maytansinoids) attached per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 3.5 2+: 0.5 drug molecuies (e.g., maytansinoids) per antibody. {002025 The anti4CD37 antibody or fragment thereof can be modified by reacting a. tional crosslinbing reagent with the anti—CD37 dy or fragment thereof, thereby resulting in the nt ment of a linker molecule to tbe anti~CD37 antibody or fragment thereof. As used herein, a "bifunctional crosslinking reagent" is any chemical moiety that covalently links a cell—binding agent to a drug, such as the drugs described herein. In another method, a portion of the linking moiety is provided by the drug. In this respect, the drug comprises a linking moiety that is part of a larger linker molecule that is used to join the cell-binding agent to the drug. For example, to form the maytansinoid DMl, the side chain at the C—3 hydroxyl group of maytansine is modified to have a free sulfliydryl group (SH). This W0 2012/135740 _ 54 _ thiolated form of maytansine can react with a modified cell—binding agent to form a ate. Therefore, the final linker is assembled from two components, one of which is provided by the crosslinking reagent, while the other is provided by the side chain from DMl.

The drug molecules can also be linked to the antibody les through an intermediary carrier molecule such as serum albumin.

As used herein, the expression "linked to a cell—binding agent" or "linked to an anti—CD37 antibody or fragmen " refers to the conjugate molecule comprising at least one drug derivative bound to a cell-binding agent anti-CD37 antibody or fragment via a suitable linking group, or a precursor thereof.

One linking group is SMCC.

In certain ments, cytotoxic agents useful in the present invention are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include esters of maytansinol and maytansinol analogs. Included are any drugs that inhibit microtubule formation and that are highly toxic to mammalian cells, as are maytansinol and maytansinol s.

Examples of suitable maytansinol esters include those having a modified aromatic ring and those having modifications at other positions. Such suitable sinoids are disclosed in US. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 020; 064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497 and 796.

] In a certain embodiment, the immunoconjugates of the ion utilize the thiol-containing maytansinoid (DMI), formally termed Nzi—deacetyl-NZZB—mercapto—1- oxopropy1)-maytansine, as the cytotoxic agent. DMl is represented by the following ural formula (111): o 91% T SH 9' \ <3 «“3. - 9x, ;« , x. - w , K .V- \ .14 \ .2- \Y \x‘ \xE-e" ' \rA:‘~.- U l K , l K , :, t 1 .=: t " 4, -\,\~/ \ ~"flA\‘L‘O_ \k, ,‘v"\\ /‘:\, \Nfi/I/ .v" N, & ‘,\" \' H" ‘ N H I O MeO (111) In another embodiment, the ates of the present invention utilize the thiol-containing maytansinoid N2i-deacetyl-N27(4—methy1mercapto—l- oxopenty1)—maytansine (e.g., DM4) as the cytotoxic agent. DM4 is represented by the following structural formula (IV): 23' \ 2: 2 C. 2222*]wa \2 3 2222\2 wa/‘l «(g-"Lu, 2, y"\\ %‘\ i \‘fig «~+:L\ i S C N 0 M90 HO H (IV) ] Another maytansinoid comprising a side chain that contains a sterically hindered thiol bond is N2,~deacetyl-N-2’(4-mercapto-l—oxopentyl)—maytansine (termed DM3), represented by the following ural formula (V): Meé (V) Each of the maytansinoids taught in US Patent No. 5,208,020 and 497, can also be used in the conjugate of the present invention. In this regard, the entire disclosure of 5,208,020 and 7,276,697 is incorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemically link the linking moiety. For example, the C—3 position having a hydroxyl group, the C-14 position modified with ymethyl, the C-15 position modified with hydroxy and the C—20 position having a hydroxy group are all expected to be useful. In some embodiments, the C-3 position serves as the position to chemically link the linking , and in some particular embodiments, the C-3 position of maytansinol serves as the on to chemically link the linking moiety.

Structural representations of some conjugates are shown below: W0 2012/135740 PCT/U82012/031648 z \r .- CI ’ ‘ \ I' o i I 2 MeO Q ,.~-"‘\\ «:3 R' E V O \f’w o ,‘3 E .J" \O E : N 0 Ab = Antibody MeO HO H '=HorMe DM1: R=H, q=1 3 DM4: R= CH3,q=2 A, n =1-24 g 2-8 Ab—PEG—Mal-DM 1/DM4 (VI) 3+ "1 o o g H N S -, i g 91"! 0 Cl Q I N/\/U\N/\,QO\\;~"“}~4\. $‘N g E i E “Jib \ “O, H ",- MeO\‘/_fl\ \{IN -.__“\\ \\ ‘5‘} O \‘a .,\ \3 {/1 O 1*” ‘\f} xi i E E". \O \“wy\\ /““‘ Fri” fiN/koV,” Ab = An‘ubody.

MeO HO H Ab-PEG4-Ma1-DM1 (VII) { /‘ Z 0 3 Med H0 H Ab = Antibody . R' = H or Me i DM1: R=H, q=1 i DM4: R= CH3, q=2 : = 1-24 K «2192-8 AbPEG—SIA—DMl/DM4 (VIII) /WMflN j k \ ‘\..»v-“' _\, k : ‘\ s = O MeO HO Ab-SMCC—DMl Ab = Antibody Ab-SPP—DMl (XI) _ 58 - f“ ’"x t O S \ \\ , O O 9' \ Et ? . a 3‘ ..

MeO\/\ N \1.7-» ,./\, w I“§ \a \\\\ Ab—A_ m1'b d0 y ”“aim/“kl,..«""\,\_,,_..._i>\, x MeO HO H “ _,.3 2-5 Ab—SPDB-DM4 (XII) e» w\ .5 SOs-N5 s i VT HMAb \8 2" x\\ , \XY}, r’ ‘O “We, ~\_ /_.a\ i k Ab=Antibody § V" ‘v-ws‘x 3 \S 5 N O : ‘ MeO HO H “a, “9“" Ab-SulfO-SPDB—DM4 (XIII) l descriptions for producing such antibody—maytansinoid conjugates are provided in US. Patent Nos. 6,333,410, 163, 6,716,821, and 565, each of which is incorporated herein in its ty.

] In general, a solution of an antibody in aqueous buffer can be incubated with a molar excess of maytansinoids having a disulfide moiety that bears a reactive group. The reaction mixture can be quenched by addition of excess amine (such as ethanolamine, taurine, etc). The maytansinoid-antibody conjugate can then be purified by gel ion.

] The number of maytansinoid molecules bound per antibody molecule can be determined by measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm. The average number of maytansinoid molecules/antibody can be, for example, about 1—10, 2-5, 3-4, or about 3.5. In one aspect, the average number of maytansinoid molecules/antibody is about 3.5 i 0.5.

Anthracycline nds, as well as derivatives, intermediates and modified versions thereof, can also be used to prepare anti-CD37 immunoconjugates. For example, doxorubicin, doxorubicin derivatives, doxorubicin intermediates, and modified bicins can be used in anti-CD37 conjugates. Exemplary compounds are described in , which is herein incorporated by reference in its entirety. Such compounds include, for example, compounds of the following formula: W0 2012/135740 PCT/U82012/031648 _ 59 _ wherein R1 is a hydrogen atom, hydroxy or methoxy group and R2 is a C1-C5 alkoxy group, or a pharmaceutically acceptable salt thereof.

] Conjugates of antibodies with sinoid or other drugs can be evaluated for their ability to suppress proliferation of various unwanted cell lines in vitro. For e, cell lines such as the human lymphoma cell line Daudi and the human lymphoma cell line Ramos, can easily be used for the assessment of cytotoxicity of these compounds. Cells to be evaluated can be d to the compounds for 4 to 5 days and the ing fractions of cells measured in direct assays by known methods. IC50 values can then be calculated from the results of the assays.

The immunoconjugates can, according to some ments described herein, be alized into cells. The immunocongugate, ore, can exert a therapeutic effect when it is taken up by, or internalized, by a CD37—expressing cell. In some particular ments, the immunoconjugate comprises an antibody, antibody fragment, or polypeptide, linked to a cytotoxic agent by a cleavable linker, and the cytotoxic agent is cleaved from the antibody, antibody fragment, or polypeptide, wherein it is alized by a xpressing cell.

In some embodiments, the immunoconjugates are capable of depleting B-cells, e.g. autoreactive B-cells. For example, in some embodiments, ent with an immunoconjugate results in a depletion of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of B-cells.

In another aspect of the invention siRNA molecules can be linked to the antibodies of the present invention instead of a drug. siRNAs can be linked to the antibodies of the present invention by methods commonly used for the modification of oligonucleotides (see, for example, US Patent Publications 20050107325 and 20070213292). Thus the siRNA in its 3’ or 5’—phosphoromidite form can be reacted with one end of the crosslinker bearing a hydroxyl functionality to give an ester bond between the siRNA and the crosslinker. Similarly reaction of the siRNA phosphoramidite with a crosslinker bearing a terminal amino group results in linkage of the crosslinker to the siRNA through an amine.

Alternatively, the siRNA can be derivatized by standard chemical methods to introduce a thiol group.

W0 2012/135740 2012/031648 _ 60 _ This thiol-containing siRNA can be reacted with an antibody, that has been modified to introduce an active disulfide or maleimide moiety, to produce a ble or non cleavable ate. Between 1 — 20 siRNA molecules can be linked to an antibody by this method.

III. Polynucleotides In certain embodiments, the invention encompasses polynucleotides comprising cleotides that encode a polypeptide that specifically binds CD37 or a fragment of such a polypeptide. For example, the invention es a polynucleotide comprising a nucleic acid ce that encodes an antibody to a human CD37 or encodes a fragment of such an antibody. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double—stranded or single—stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

In certain embodiments, the polynucleotides are isolated. In certain ments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a sequence ed from the group consisting of SEQ ID NOsz4—l20.

The invention further provides a polynucleotide comprising a sequence selected from those shown in Tables 7—10 below.

Table 7: Variable heavy chain polynucleotide sequences mmVHPolvnucleotide Sequence(SEQ_:IDNO) §mUCD37-3 3°gtgmagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactg tctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtg gctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaag aaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtact Iactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgca (SEQ IDNO: 121) {fichCD373 aagcttgccaccatggctgtcctggcactgctcctctgcctggtgacatacccaagctgtgtcctatcacaggtgcaggtg aaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccac ctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcac . tcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtz? ctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggg itcacautctct*cagcctctacvaam‘wccc(SEQIDNQ: 122) huCD37-3v10 aagcttgccaccatgggttggagctgcattattctgtttctggtggccaccgccaccggtgtgcactcacaagtccaagtc 1““ caagaatctggtccaggtctggtggccccttcccaaactctgagcatcacctgtaccgtttctggttttagccttaccacctc itggtgtgagttgggtacgccaaccacccggtaagggtctcgaatggctgggtgtaatctggggtgatggttccacaaatt accatccttccctcaagtcccgccttagcatcaaaaaggatcacagcaaaagtcaagttttcctgaaactgaatagtctgac cgatacagccacctactattgcgccaagggtggttatagtcttgcacactggggtcaaggtaccctcgttaccgt: umnniflctcctcactagtaccaaz.fgeccc(SEQIDNQ:I23} __ j t huCD37—3vll aagcttgccaccatgggctggagctgtatcattctgtttctggtggcgacagctactggggtccactcccaagtgcaggta caagagtccgggcctggattggtcgcaccaagccagaccctctctatcacttgtaccgttagcgggttctctctgacaacc Gtgfaztgagttgggtga=x(cagccaccaggaaagggactxtiagtggctgggggtgatttg 2* g W0 2012/135740 PCT/U82012/031648 _ 61 _ : atcaitccagtcfiaaatL’nggtLGtLLattaaz.c1d&z,dCCZ€idLId.idfCtCfiflngL1' aaactcaaLagLL. cOcafiacactgaacgafiactfiLgLLaaaHUaLL sjaLLLivtLaiLLgcatcazicaaa L.c{SEQ 113‘ N(J:1 "’ V..LLLL.L“....... 777777....LLLLLLLLL““““.._. 5...“...«n 12 EcagamcagiL‘tggtg agtctggacm anLIgaagdagLLt‘ggdgq(magma... U Lfictgggta‘tacattcacaaagtatggaaigaactgggtgaagcaggct'aamadawvtfiaaagtg L'tga"{*aafiLngggdLagrtttgcctic 1 gatgggctggataaacaccaacactggagagtcaagaaatgc 3 tctflggaaacctctgccacvcactrILLi‘att‘LQcaLzatLaaLaLLLEL:aaatatgaggacacggctacat E2:131nggaaggggL-LgtagLLgLLoaLtggngcaaLLdLLaciLtLaLagtLtLLLca (SEQID 3.....L....,,_............................ . .1 VVV........ .LLL..LL........

V........... .LL.............._._................ gchCD37- 12 aagcttgc3LLatggg-aogiLaioca‘aaiLLLL'tiLtgotcgctaLtuLmLLggt9:91.LtLaLaLaitLaa1‘91; ttcaaagiggccmoathbaoaaagLagggaaaoatwaaadtaathLLaaggca mggfiacactficacaaa cai(53.81;{9.331033%amcccagggcaagf “{LLLadana‘iga0'{Egg}3'9:LaaiaccaacactcDgazg agtLtL-iwaatLLigavgaomaaggpccwffloficwcdggagaca'w gccagcac.LagLLfiacctgcaaafi aaLaatLtgaagtatgaEgltdgdgfldfiLCLfimGEGCgELNngLac-tgLLgfigLLagactggggacaaggtacca ‘ act);m‘cLaLLva3:caL{$12.00LL (QLQ 11) N(J:126}"3v" “W.L........._.‘“............ ... ....VVVV.V.....................L L.,a Digcagm4337335333,:«Ctgat.CtgéfigdadcfimkdfitLLttLLaLmaLLLgL (3O teactggctactccatoaccagtgamiggctggcac.tggaicLLL”castfiLLaOLaaaaagctga3'0 atggatggcctacaiacmiacagig.{giggcacto6LtaLaaLLLathLtLama‘rtLULLatL‘"Ltac aLILLvagacaLfi‘“ acaaccam‘tLttLLLgLfiofigfik1gt£VaLv1chiJaz§E~Y’lcaC&gLcacai afiactgtgcagdrwcLaLtaLULUacgg{VLLtogmUmscwgwcLaaggactctggmac l”i?§s:33f:§.<.§fl;11}.3§91” _...- _________________________________________________ .,,,,,,,,H,..”,,......,,..................................___.._chCD3738 E aagcfigccaccatg gciggag‘itgiatcafictgmfio3190mmLGLLLLLiWaLLLLatLLLLLaotLLaaLtLL aggaatctggccm‘gacctacttaaUL‘VLiLLcagaO'cLILrccctoaLLTgLameLagbamLicaatca'L‘aiLLag octfigt‘Ltgficac‘woatcasmaatitLLLgoLaacaagttggviatogatgthiacafictgtatagngjgggtaccg 5 1:) attacm L‘Li Lthcaaga9ccgaaL‘zMatLaLLai—Jf(rataLawLacLLaa-‘caamfitctm QLLtcag{(2:ngt: acia"°ga.dg..3.iacgcta.taLtattgtPLLagmLtaLtaiggaiat’rgfiLdtLguLOELLLthggggccaggga acccm‘tagggg34mg. v; ' a ' V ;, 33) . .7.....L...LLLLL“3.““....L..... hUCB37'38 aagcétgcc' “3&th,gtigga'5 ctggzLiLLdLiLaLaogtLLangc aagavtLLguicgmguggaaacccaocLaLtLLcTL.gtLtLaLLLLmeiLt.LLigthaLtLtmaLcagtgg1&1").

Ltgvcafiggmmggwg‘mcLL‘9«{taagvggctggagto93*ggbLatatatLLt9faLaUL3332123933"c gai‘tacaaLtcL.agtctgaayaficafiga{€304.4thcQLLLLaLawLaaaaaccafit’rmLLnogthgCtsigi . : 5E?.3.’*d0LtgagaLaLchLtaLnaLtafiotngtggmLidtgngtowLLIggmgtgtafivqggacaaw ’ ‘ ' ‘ “c @331955‘foHJNQ: 179) _ » -_. muCD37-50 ag gLagLfiLagg gt aggucdgacLiofiganLLtttLaJLLdLmLaLtLaLctcactg {LaLWOLtaL:CLthcaLLagtagmchtcC,L'1L£6gatLL69agttmaggaaacaaaaégga atggatggga.taLaLaLtL{110391353918gLaLtgthLagLLLaitctcaaaagt-c.gaatctctam gacactLLaaLaaLLaLLtLLiLLLLLagttoaartL-Lfih'acgamaLaLagLLde‘:(O g .......... afiaLtngaagwggmLLaivgitaL‘LLLgcLtggmgcfiactggggccaagggaLLtciggtLac L.:3:3:ct:,_..:ca<83“ ..“a””,.V,..,..,,,,..‘,.........................................'Hii‘éfié'ifimw 33»ngKELCLlLLc‘l‘nggbgig’kaLOLataatLLthLt gtigciactctaccvcavtccafi'VL'wggtgcagctgL figgagtccggCCCCi‘flOL-EULiCddUL‘LfiL-RagagtLtgaotrgacfiEL‘LEgmLWLLMLaGLtagccagcg gmcgcfiggLaLfgLLfiLagaLducatLLwLLaaLaaacgflagtogatog&atacdmtLtaLE’LaLOcLLaaLL g:ctattchthcLLtgaaatnccggatcacmiaLccgtL33:10acmtaagaas.LaWLtQLachtgaaLaflLvtt' L<{Lagugacamocam013k1:81.{EEGCLngjLUd'LéifldU’gLitnggdgcfigvfLthtacthggLLad90 ' 5CflCC-G muCD3751 gatgigcagLtléflggfigtLfiboakabchtgfiammHLLLaL‘LLtuLaLiLa.LEgLaLtg3 IcactggciacLieaLLLLLangmLOLLEggLacfggamggagiticcaggaaacaaadgga 3:333«Egg,thaLstaLaLtdLaUELLagL-aLtaaL‘taLaocLLamNcaaaagtcgaatctctam actsg2agactcatLcaagaaLLagficficctmaE,ttLaaLtLtLLLaLtdLigaagaLacagccacat E gcaagvawaiaaatgvgmcngLLt9.j€ttLina.[Ugggccaagggactciggtcac “L La ssmmxo 133) AagcttgLLaLLaLngLLLLLLfiLLatLaLLLtgmLtggtggccactgwacirm " ,L LLLaOGaaichf-L:1321"“C(- try! (n \ , »'\ L- 4 (L ., PCT/U82012/031648 _ 62 _ W1”ggctttgcttggcactggattcggcagtttccaggcaagggactogaatggatgggctacatccattacagtggctcaac caattacagccctagcctgcagggccgaatctctattaccagggatagttctattaaccagtttttcctgcagcttaattccgt gactgcctctgacacagcaacttactattgcgcccotggctactacoggttcOgagcctgtttgtatactggggtcaggg muCD3756 gatgtgcagcttcaggagtcaggacctgacctogtgaaaccttctcagtcactttcactcacctgcactg tcactggctactccatcaccagtggttttgcctggcactggatccggcagtttccaggaaacaaactgga atggatgggctacatacactacagtggtggcactaactacaacccatctctcaaaagtcgagtctctatc .3 gacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaa gaggctactatggtttcggggcctggtttgcttactggggccaagggactctggtccc Lemmag3SEQ1D NQ:13332)33 h CD37—56 aagcttgccaccatgg0gtggagctgcattatcctgttcctcgtcgccaccgcaaccggcgtccactcccaggtgcagct : gcaagaaagcgggccaggattggtaaaaccttcccagtctctgagtcttacttgtaccgtatctggatacagtatcacatct ggcttcgcctggcattggattcgccagtttcccggcaaggggcttgagtggatggggtatattcattattctggaggtacca actacaacccttccctgaagagtcgagtctcaattaccagggacacttccaagaaccaattctttttgcagcttaattcagtg accgctgccgacaccgctacttactactgcgcccgggDctactatgggtttggtgcctggttcgcctactggggccaggg -57 gatgtgcagcttcaggagtcaggacctgacctgfigaaaccttctcaotcactttcactcacctgcactg i tcactggctactccatcaccagtggttttgcctggcactggatocggcagtttccaggaaacaaactgga atggatgggctacatactctacagtggtagcactgtctacagcccatctctcaaaagtcgaatctctatc 33 actcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaa actatggttacggcgcctggtttgcttactggggccaagggactctggtcactgtctctgca (SEQ ID 311L133.) ___w huCD37—57 gccaccatgggctg0agctgcatcattctgtttctggtggccacagcaactggcgttcacagtcaagtccaactg caggagagcggccccggactcctgaaaccatctcagtcactcagtctgacatgtactgtgagcggctacagcattacctc aggcttcgcttggcattggatcaggcagttccccggaaaaggtctggagtggatggggtacattctgtacagcggcagta cagtgtattcaccctccttgaaatctaggatatcaatcacacgtgatacaagcaaaaatcagttcttcctccagctgaactcc gccgcagacacagcaacctattattgtgctcgcggatactacggatatggcgcatgttcgcctattggggcca ggggacactcfituaccgtttccrgccocctccacaaaLiLCCC(SEQIDNQ:135__)_\v. \:_____ ____ .1 252-3 gaggtgcaggtggtggagtctgggggagacttagtgaagcctogagggtccctgaaactctcctotgcagcctctggat tcactttcagtagctatggcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaaccattagtagtg gtggtagttacacctactctccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaaaaccctgtac ctgcaaatgagcagtctgaagtctgaggacacagccatgtattactgtgcaagacatagttactacgatactagcgtcgac tactggggtcaagaacmiségcgtctccwMSEQlDNO182) 3 Table 8: Variable light chain polynucleotide sequences 11"“""1 l VL PolxnucleotldeSequence(SEQ1D NO) 1 muCD37-3 gacatccagatgactcagtctccagcctccctttctgtatctgtgggagaaactgtcaccatcacatgtc E gtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcct E ggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacag tattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggta ‘1fl1x“WWWMMA_uHA“Euctaca1‘{ft/acattcggtggaggcaccawctm‘aaatcaadcct(SEQ112NQ:136) i3rchCD37-3 gaattcgccaccatgagtgtgcccactcaggEcelggggttgctgctgctgtggcttacagatgccagatgtgacatccag atgactcagtctccagcctccctttctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgca gtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgtgcaacaaacttagcagatggtgt 1 gccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttggga E cttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgtacg (SEQ ‘ 333311) No 137) _ huCD37—3 gaattcgccaccatgggt‘tggtcctgcatcatcttg‘tttctcgtggccacagccaccggtgttcactctgatatacaaatgac (1.0 and 1.1) tcaaagcccttccagtttgagcgtaagtgtgggtgaacgcgtaacaatcacctgtaoagctagtgaaaacatccgcagta T1,”,Mflflflflwu. cttcactattctctggttcaaottcg g_maccGattattcacttaao’ttcaactcacc:t accagaacatttcgcctacatatta ictgtcaacaactactgg«4.1cgtacgacctggacattcggtcaagrtactaagccggaaatcaagcgtacg {SEQ 113 WM) 1 muCD37 12 gacattgt caccctcctgc151;cttaotgtatctctg fiwccc'wccaaactcagta‘a tacgatact‘atttgtactggttccavcao'Laaaggacagcc fiacccaaactcficatcaactatgacaacxtamatctggggtca'V°caggttcagtmcact{2g tctgggacagacttcaa‘cccaacatcatcctotggaggaooagcatactgcaacatattacwtcaac ' " iacac‘1tO_gaooattccgtacacattcggacgcgacmazctggaaataaaas ...........................g. chCD37-12 oaattcoccaccatgc"ttgvtcctgtamacctOEtctcggtgcancccractgccgmatactgatattctacis:act005 gtcaccagccagtctcficamgtccct00occ'tgcgtgcaccatcccctccccogcctcacagtccUtgagcac1a gctcttattcctatctctactgctttcaacagaagccawaagcc‘ctacgctgctvatcaaotacnctccaacctcgc caccgmcttcccgctagattctctggttccggtagcUgaact’satttcactttgaacatccacccccttgaggaaagga tacc ccacttacéattctcaacactcttgaoacattccttaacctttgcaggaogaacaaagctccaaattaaacctacg 13:5ch . .». 1 muCD37—3 8 caaattgttctcacmagtctccagcaatcatgtctocatctccacgggacaagotctcacctgca gct‘aao'tgtaacttacatmactovtaccaagaagtcawcacct aaaagatggat ttatgacacatccaaactcocttctggaotccctmtcccttcactYfY’ggtUggtcgggaacctcttac tctctcacaatcaocascatggaccctcaacatcctcccacttattac1gccagcagtgP‘xz rgattactaam i ‘ cacccac: .tcc ‘ c’vgaccaagctgcaaattaaacc) (SEQ 11) ___..uu_............"“......____________________ “c“ \<“““ chC1337-38 gaattc; caccatgcgctggtcctotattacctgtttctcctgcccacaociataggtgttcattctcaJ L .ttgtgctgac accavctattatvtccgtagcccggccagaaagagacaatfiacatctaowcta01tcttctgtgacttacat gcattggtatcaacagaagtcaggtaccagtcttaagcgttooatcwccacacatccaaactcxtcutccg.. CC? agtccctg cagcggaggtgggtc.ccaccagttamac'gaccatatcctct'ttgmacctcaacawctoctacitatta ttgtcaacaatggatttctaaccccccccaccttiggtggngaacaaagctggagatcaagcgtacg (SEQ ID N132142) : gaattcgccaccatccvv{gotcctgcattattctgttcttggtcgccacto1tactcccgacactctattctvctcacaCy "unannflflflrflwr . E c 311“debkfiulgfcLUCt'CCCCGEEZQ1gEUC(milk*c'ttgtcatoctctoccagttcctcg:.gacatatatgc ~ attwtatcaccaaaaacggtacctctccaaaaaatggatcctacgacacttcaaagcttowtaggcgttcctgcca t gattttccgggtctgggictggcacttcaacagtctgacccttwtt’catgaancivaagacgca‘ccacctattactgt t .\ t cagcagtggatttcaaatctccaccitcggcggcgyaacaaactggagataaagcgtacv {8131:1113 ‘ w NO: 143, caaattcttctcacccagttccagcaacatgtciocatctxcac.g ggacaaggtcaccatgacdgca gtgccacctcaagtgtgacttacatgcactgstaccagcagaagtcagg;acctcccccaaaagatggatuatgaca"> 1,.V......_________ [ccaaactgccttatgagtccxtggtcgtttcactggtacgggtctgggattactctctcacaatcagcagcatgg antarctoaagatoctoccacttattactgccaocao‘rgwagtoataacccccccacgttcggctcggggacaaagttgga {3EQMID N01143: Wmflflfl,flflfl,”,, ccaccatgccacfi“‘atrtcattattctctr‘tugttgctacgmaggagtacatag oasaagtccicac gcattcgtatcaocaaaagcct“occaatcccctaaaagctgga‘tctaccatactttta'atccocataccctgtgcccgc aafmttctcongagtgfigcagtogagcacrtatagtctcaccatcactiaatggaagtagaogtgcagcaacctatt acctcaocaotgvt"gataatcccgctacttttggtcagggtacaaaficumwattaagcctac ( ‘EEQ 1D 0:1/1_‘?_:__ muCD37-5 1 caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgca gtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacaca ctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcaacatgg aggctgaagatgctgccacttattactgccagcagtggagtagtaacccacccacgttcggctcggggacaaagttgga am(SEQID149146),,,,,,,,,,,,,,WM 1111163637351 . t gaattcgccaccatgggatggagctgtattattctgttcctggttgctactgctactggcgtccattccgagatagtcctcac t ccagagccccgcaaccatgagtgcctcccctggggagcgagtgactatgacttgttccgccacttcttcagttacctatat \ gcattggtatcagcagaaacctggacagtctccaaagcgttggatttacgacacctccaacctggcttcaggagttcctgc 1 taggttcagcggatctgggtctggcacaagttattcactcaccattagttccatggaggccgaagatgccgctacttactac 1 cagtggagcagcaacccccctacattcgggcagggaactaagctggagatcaaacgtacg (SEQ ID Lcccap—Magcauuu. ........"v. ___NO:14_7) W0 2012/135740 PCT/U82012/031648 _ 64 _ muCD3756¢ caaattgttctcacccagtctccagcattcatgtctgcatctcCaggggataaggtcaccat9acct9ca gtgccagttcaagtg?acttacatgcactggtatcagcagaagtcaggcaoctcccccaaaagatggatttatgacacat CCaaactggcttctggagtccctgctcgcttcagtggcggtgggtctgggacctcttac tctctcacaatcagcaccatggaggctgaagatgctgccacttattactgccagcagtggattagtgacc caCOTtCMaggg ggaccaaoctggaaataaaacm(SEQIQNQ:__148) ..

E 56 gaattcgccaccatgggctggtcct tatcatcctgtttctggtggcaaccgctactggg ttcaCECtatattgtcctgacg g g E : E acagagtccagccttcatgagtgcttctcccggagaaaaggtcacaatgacttgttcagcttcctcctccgtcacatacatg E cattggtaccagcagaagcCtgaccagagtcctaagaggtggatctatgatacaagcaatctggcttccggtgtcccctc E ccgcttttcaggcggcggaagcggaactgactatagcCttaccatctcctcaatggaagccgaggacgctgctacatatt E E agcaatggatcagcgaccctcctactttcggacagggaacaaaattggaaattaagcgtacg (SEQ ID muCD37—57 caaatt9ttctcaccCagtctccageaatCatgtctgcatctccaggggagaaggtcaccatgaCCtgca Egtgccacctcaagtgtgacttacatgcactggtaccageagaagtcaggcacctcocccaaaagatggatttatgacaca tCcaaactggcttctggagtccctchgcttCagtggcagtgggtctgggacctcttactotctcacaatcagcagcatggE Eaggctgaagatgctgccacttattactgccagcagtggagtgataacccacccacgttcggctcggggacaaagttgga E ‘CmfSEQ ID NO:150) E huCD3 7-57 gaattcgccacCatggggtggtcctgtattatcctgttcctggtcgcaaccgccacaggcgttcactccgagatcgtgttga 1 ctcagagcccagccaCCatgtCCgcttCCCCCggggagagagtgacaatgacttgttccgccacaagttctgtaacctac atgcattggtaccagcaaaaaccaggacagagtccccgtcgttggatttatgatacctctaacctggcttcaggcgttcctgE CCcgcttttctggtagtggatctgggacttcctatagccttaccataagctctatggaagccgaggacgccgctacatacta ctgccagcagtggagtgataacccccccaccttcgggcagggaaccaaattggagatcaaacgtacg (SEQ ID No912 _I. 252-3 E gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttmagggc 53 aagtcaggacattagcaattatttaaactggtatcagcagaaacCCgatggaactg’ctaaactcctgatctactac acatcaaaattacactcaggagtcccatcaaggttCagtggCagtgggtctggaacagattattctctcaccatt agcaacctggagcaagaagatattgcCacttacttttgccaacagggtaatgcgcttccgtggacgttcggtggE aggcaccaa9ctaactcaaacgg (SEQ ID NQ: 183) Table 9: Full-length heavy Chain polynucleotide sequences Antlbodx ~ HeaV ham cleotlde SC}:EuenCC (SEQID NO‘ E ___.“ __ __enfth ChCD373 aagctthCaccatggctgtcctggcactgctcctctgcctggtgacataCCCaaoctgtgtcctatcacaggtgcaggtg aaggagtcaggacCtggcctggtggcgCCCtcacagagcctgtccattacatgcactgtctcagggttctcattaaccac ctctggtgtaagctgggttcgcCagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcac aaactatcattcagctctcaaatCCagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagt ctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctgg E tcacagtctctgcagcctctacgaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacag Ctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgt gCacactt‘ttcccgctgtgttgcagtCctccggtctgtadcactgtccagtgtcgtaaccgtcccttctagcagcttgggaa CCcagaCctacatctgtaacgtcaaccataaaCCatccaacacaaaggtggataagaaggttgaaccaaagagctgtga taagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcCtgtttccccccaaacccaag gacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaa ctggtaCgtggatggagtcgaggttcacaathCaagaCcaagcccagggaggagcaatataattctacatatcgggta gtgagcgttctgaccgtgctcCaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaaeaaggctcttcC cgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctaga gacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggag tctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactcca agttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaathatgaggctctgcacaatcacta iaéf W0 2012/135740 PCT/U82012/031648 ., 55 _ LtggtssLatgtstkgt;{90LsLtTsLaaaLtLtriavsaLLasLtgtaLsmstqgtfiLagLst‘taLeaLstL ng‘tgt*aottgggtL'tsgse'assetsLngtaagggttsgaatgfisiLgg:gtaaktggggtgatgg‘tsLaLaaa‘tt assatsst‘tssstsaagtsLLgLLttaOLatLaaaaaggatsasagstaaaagtsaagtfitestgaaaLtgaatagtstgas agsagsLgatasafisLasLmsLaLtgLLLL‘aaopaget‘tataotLfioeasasingtLaaggas:sstsgfiassg stsstsagstagtassaagggscsatsaotLttsLLLttgo'iLsaaLrtstaatssasaagsggtggaasagstgsast ggga'n'gLLgt‘taaagattattzsLstgagsstgtgasagtgagLtggaatagsggagsaflgasttsaggtgtgeasLa fittcsLscgstgtgttgsagtsstssggtstgtastsastgtssagtgtsgtaaesgtsesfietagsagsttgggaasssaga setasatstgtaaegteaassataaassatssaasasaaaggtggatmgaafggfttvaassaaagaostotgataagasasatasaigssstssttc ’tsstgsacsagavst"sggaggtL‘LatstofgttLstLtttsLsLsLaaaLsLaaogasast sttatgatststsgtasLLsaf‘aggtsaLLtvtgttrttfisgasgtguossatoaaoatsLsgawtiaaafisaaLLgta L4UtggatggagtLgaogttsasaatgseaagasaagesszngaggamaatotaaustasatahngmgtLao" OttLtgasLUtthLsaLLaaattggstsaatggaaaagag‘tasaagto(aaggtgtcLaasaagthLttsLsgLtss auto«gmaaLtatLtLLaaaLssaaagggsaossangcaaLL"‘f3gigtataeat‘tgssescatstagagasga 0sigaL‘saaoaasLaggwagtLimLagistggtLaagggnttLtaL‘L‘ttstgasai‘tgstgtagag‘tgLgaUtLtaaL ggasagssaoaaaasaastasaagaLaLtLiLL‘LLLag‘wstgoaLageUasggg'=g7-'L‘.tts’ztssEL‘tastssaagttga . LtgtagasaagtLtagatggLagLaaggaaaLLtLttctsLtgL‘tLagtaatgLatgtgLsetigeasaatsactatasss : LaL a{essay LLHag (SEQ ID N0:33)1:LL'.LLLLLLL.4\:L LLLLLLLLLLLLL“ __......LLLLLLLLLLLLLLH LLLLLLLLLLL.QM-elh huCDS ”3% 1 L L aaosttgLsasLaiLmLtUgathomiLattstgfitstggtggsgasagstastg<3LgtsL34Ltsssaagtgsaggta L saagsgtseggLsthafttggtsgsaLs'agesagaLsststLtatsast‘tgtaLLgttagsgg.magasaase 3 agtogax‘rgao{tagg‘tgaggsagssassaggaaagggastggaGiggstgggggtgatttggmsosogeagsa i aaaLtatLaaLLag‘LLmaatstsggitgtssatLaaaaaao22LsataotaaatLtsaagttLtLstg'LaasELaatagsL4t . 1 gasagssgsagasastgstasgta'taetUsgssaaawaoLataLwtLtggctsastgt‘tasaggggawsiggtgassgtgtsatssgsateaasaaagggLseatLagttitLLLLELLULLLLaagttsLaaotsLaL'taLsggtggaaeagstgcactgggatgsstLgttaaagattatttsLstwTestO’tgasathaaLtm‘hataggggLattoa"ttsa0giLtosasasttttsLsthgm‘iLLant"‘tLLLoILLOXasXLaLtgtLLagiLtegtasgt‘tstLLagsaws‘LtgggrxLLLagaLLLaft‘taamtcaasataaaLLatsaLar210n“taagaaggt‘tgaaLLaaa'raoswtga L taagaeaeatasatgLsL‘iessttgtssttgea:‘agag‘tsstsg‘gtsLa‘isigtottsLtwmsLLesstaasssaag aLaLtLttagateteisLgtastscagaaag tLgtg,tttg’ttgtLfigaLLtLaLLsatoaagatsssgaggfiaaatsaa . csaagsseagggax'gageaatasaattstmatatsgggta : ggtaL4stggatggagLLgaoUttLaLaatocLaag gtgagsgt‘tLtgasLgtgstssaLsaagatflthLaatggaoagta'm‘gtg‘aaggtgLL‘2a‘saaggsLtsttss sgsta‘:LLattgagaaaaLtatLtLsaaasL4aagfiggsesaLL‘gggaascsLaggtO'tatasatV—VLL.L‘a‘tstaga gasgagstgassaagaasLagatoagtstsasttgtggstaaggggtttiaLsLttsMaLa‘tthgtaga'Itgggag .4 tstaasggasagssagaaaaeaaLtasaagaLaasrsesesagtgLtggaLaosganggaosmttststaetesa . atwttgastotaoasaantiwarW‘LgsaaggaaaLgmstsLtthsugiLawLagaggctctgLasaatcas‘ra L L {LLLLLagaaatmLt tgastsgggxg 0 ED NO:E34) L V V __V_ a * aa;7“ft0sLaLsangY'thgtsa’tgLataatLstsmLtggtsthaLtaLwLsggigtosasLLaLa'Ta‘a‘Lathgg t shCDB’Y—lZ ttsaaagtggLsLagagLtgaaaaagsLaggggaaasagtgaaaataagitge:aggsa‘zssggttasasmsasaaa gtasggsatgaastgggtsaagsagvsssaGggLaagggBLtLaaatwatoggtg;at‘Latassaasastggsg agtstaggaatgsigaggagitmagggLs'igtttssst‘LLavestoga0aLaaUtgLsavsasavsfiasstgsaaats i } aasaatstoaaotatgawatmagsaaLetatttefigs0gL:sgLggca01LmfisaUaLtggovasaaggtaesaLsagaLtg‘ia‘LLLagtgLsagsastaagLssafLathisst‘tgfgen.Laagttstaaatssasaagsggtggaa I sagstgsastgggatgsstegfiaaagattatfisedgageLtgtgasagtgagstggaatagsggagsa‘ttgasttsag . , . ‘ gtgtgsasaemisssvstvtgttgsaotsstesggtstgtasteastgtssagtgtsgtaasegtessttstagsagsfigg .

LLL............... L. . a2asstasatdgmasusaasataaascatesaacaeaaaggtggataagaaggttgamsaaagagst‘g tga‘taagasasatasaz‘.gssL.4ts4s tgtLLLgsaseagagstsLtsopaag‘tsatLtgamLetgtt‘tssssssaaassc aggasaststtatgatststsgiactssagaggtsasetgtgttgttgtegaaLsgtgagLsatgaagatssegaggttaaati Laastggtasgtggatggagtsgaggttsasaatgseaagassaagsssagggaggageaatataattstasatat‘cgg LW«,.~,,,..4,””,.,.:.:.:.,............ gtagtgagLgastgassgtgctssaesaaga‘ttggstsaatgoamafiagtaeaagtgmagvtgts't'iasmogststtsssgstsssattgagaaaastatstesaaagssaaggggsagsLLasgggaasLsLaggtomfaLatigsseatstagagaegagstgassaagaassa7gbagtstsaettgtstggtLaaggggttttaLsLttLtgasattosagagt.gggagtstaasggasaossagaaLaaL'qstasaag 2:422astsLsLsagtgLtggasagLLaLngathtsttsszctast"‘aavtiLaLtLlaaLaagtstagatUUsaoLa'tggCza'tsgtt;LtLstoLisaotaatosatgagthLtgLasaatLasmtasssat‘aaatsasLLthLttagLsLags; ‘ (fit-1‘3.”{W0 11) NO: 15 WO 35740 -66— EchCD37—3 8“ ““E“‘a;gefigeggaze;giggg51gig‘Egga;5Migft’aiggt‘Eggs;a;Eggagem‘eaagigeaaasa""g; aggaatctggccctgacctggttaagccatctcagagcctctccctgacctgcactgttacaggatactcaatcacatcag gctttggctggcactggatcagacaatttcccgggaacaagttggaatggatggcttacattctgtatagcgggggtaccg attacaatccttccctcaagagccgaatctctatcaccagggatacaagcaagaaccaattttttctccgcctcagctctgtg actaccgaagataccgctacttactattgtgccaggggctactatggatatggtgcatggttcgtctattggggccaggga accctggtgactgtgagcgctgcctctaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggt ggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgact Etcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcag E cttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaag agctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttcccccca aacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgqgg ttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctaca tatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaa ggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgccc ccatctagagacgagctgaccaagaaccaggtgagtctcadtgtctggtcaaggggttttacccttctgacattgctgtag agtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcc tctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgca huCD3738 aagcttgccaccatgggttggagctgcatcattcttttcctggtcgctactgcaactggagtccactcacaggtccagctgc ccggtcctgggcttgtgaaacccagccagtccctcagtctcacctgtactgtctctggctactctattaccagtgg gttcggctggcattggattaggcagtttcccggtaaggggctggagtggatggcatatatcctgtacagcggaggaacc gattacaacccaagtctgaagagcaggatcagcattacccgggacacaagcaaaaaccagtttttccttcggctgtctagt gttacagctgcagacaccgctacttactattgtgctcggggttactatggctatggggcttggtttgtgtattggggacaag s gcactcttgtgaccgtgagcagcgcctcaacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcg E gtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattg acttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagc E agcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaa E agagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccc caaacccaaggacactCttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccga E E ggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattcta E catatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaac aaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgc ccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgt agagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttctt cctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctg cacaatcactatacccas;aaatcactgtcccttagcccagggtgactcgag: (_SEQ"I_D_NQ:157) E-huCD3750 nuuuu...mu"......mmx-"z‘"nu..."w...w.

\\\\\\\\\\\\\\\\\\\\\\\\\\Wu—hw aggagtccggccccggcctgctcaagccttctcagagtctgagtctgacttgtactgtttctggctacagcataaccagcgE gtttcgcttggcactggatcagacagcatcccggcaacaaactggagtggatgggatacatactgtactcaggctcaact gtctattccccctccctgaaatcccggatcagtattacccgtgacacttctaagaaccatttttttctgcagctgaacagcgtt accgcagctgacactgcaacctactactgtgcccggggatattatggatacggagcttggttcgcttactggggccaagg caccctcgtaactgtgagtgctgcttccaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggt gctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgact Etcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaocgtcccttctagcag cttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaag agctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttcccccca aacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgagg ttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctaca tatcgggtagtgagcgttctgaccgtgctccac'caagattggctcaatggaaaagagéacaagtgcaaggtgtccaacaa ggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgccc agagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtag agtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggaocttcttcc t3:CCttagCCC huCD37—51 E; 3:5 I ggCttthttggCCCtggafiCWcavu‘tCC210th2121310bg2‘3C3tgiatvgdtgggCtaCatCCattdCagto0th23C. caattaCabCCCtagCCth2tggchgiatCtCt2itt21CCa2gataowtts:attaaCCagtttttCCta"~;,Ctt23312tcC9C.’ f CctCtoacacama21CttactattvcgCCCUthCtdCtan.1CooattCCtwtttfitdtathggt'102223 caCCCthLCatgtctcagC chctctaCCCagC,gCCCratcajC,[titsCCCt:-.gg3..3CCdagttcta2123mmCatwcwt ggartcagC‘thCCtgggathtctC‘2gttaaagattatttCCCtgagCCtgtgacagtgagctggaatagszggagcattgaact; tcaggtgtgcacaCttttCCCgctgtgttgcagtCCtCCO‘gtCtgtathqctgtccaototcUt‘)accgtcwttctaficag CttgggaaCCcagaCCmca[CtgtmtcgtcaaCcataaaCCatCCaacaCa'ta‘wtgantagaagflttmeC21mg 2131:0thgfifaagfi-‘JfiCat?!{SaigCCCiCCfigKUECEICC$2ivtCCtCW-raggtCCat ”tottmtvtttCCCCcca aacccaaggacacEcttatgatctctcgtactCcagaggtczwtgtgttott CgaCng‘ioCCaI‘FaitgatCCCgng 3 ttaaattcaactggtacgtggatggagtcbItogttcticaatt.23233g2CCC412ngccagggavoagCaatCtdattCtaCa 3 tatcgggtagtwavcgttctgdchthtCCaCC'ta2tt0gCtC312ttg22t312331‘2331,32:23.10tgc2taoptC‘tCC2t'xsd21 3 ggctcttCCCCgCtCCC‘a‘ttg.agaaddttatCtCCaa'thcaaggggCcagCCaC;’gfidLCLCEi};jtgtatamtthCC 3 aC'tga“0210:1033.caagaaCCaggtgagtCtCadtfitCtggCC3121gg0Ctttt21CCC:tts:tgaCattoctotag 3 agtaggagtctaacggacagCcag21212123C21aCt23Ca31g2‘3CaaCtCCCCC.gtgutggacagngngbgCttcttCC. C tCt31CtCCaagjtgactstavacaagtctagatgngagC:a2:maaaCC‘ttttctcctgctcaataatm:atgaggCtCttha . 3 ‘ 3 C2121tC23C 21312333203: ‘taaatCaCtsztcscttax- act EEEQ 11} N31):t."‘3,3m 3 "huCD37—56 3 aagC‘tthCC‘CCt,gggtggagcthtttatCCtgttCCtC{CgCC’d-CCQCédCbnggtLLdCtCC(23100tf’C‘a‘FCE 3 : 3 aagcgggCCaggattggtaadm/CttCCCdCtCtCtoaUtcttacttgt1CCUt31tCtggammgtatcacatct 3 ggCttcgcctggmttgcattCUCCCCtttCCCgoC212tgqucttomtg-Vamp?gtCtCttthttCttCt 3 _ ngtacca 3 aaC:ccttCCCtgaagagtC9.21gtCtCaat‘taCC§,E,ded(,fikcaagaaCC‘aattcttttt.gCagCttaattcagtg t :1CQETCCULC. 021‘thng-tacttactactpcgcccgC,ggCt21Ct2tt3 igtttggtgcctggttcb7CCta.CtggggCC-2tggC—r UaCCCtggthCCgttgCtCC.gCCtCCamaaggWC‘atCattttCCCCttCCCtCC3121CttCt32‘321tCcacaagC tCCaavmtctgca.gggatoCCtCott2t21gfittattthC‘tgaCtgtvaCacwagtggadtdfiCCgaCCattCaC tthrIgtotgcaCacttétc"CovtotgttCC21»*CCCtCngtC.tgtz1thatothaotCtCCtadCgtCCCttCtzth2233:, Cttgggaa»;‘CC213123103:moatctgtaantC-aaccat212121CC21‘tCC2331C11C‘21aagotgoat’taoaaggttfiaaCCaaav agthtgdtawacaC‘ataCztthCCtCCHf‘TCQT‘T”aCCuC,"'iCCthUdg, C2 otgttCCtot‘tCCCCMa 2323CCCadggaCaCtCttCtgatctctCotCCtCCagaggtCaCC‘tCtUttqttgtC322Cgtg C“-W2312102131901»agg ttaaattcaaCtgntaCGtggmggaflwa'iCttcacmtgcm023C021m.CCCoggL,magcadt31tC23ttC.2tCa tatcgggtagtoaocgttCtoaCCgtUthcaCcaagattv0CtwatggaaaagagtacQagtfiCdagCtgtCCaacaa 2 gCtCtthchtCCC2ttt-1omaact'ttCthttaavccamgchthCMCCCg31CCCC2‘383‘t0t‘1taC23tth“ sactCtagmaCtzagCt1221mm210232103.aggtvagtctcm‘t‘rtctvgt2121523,?>ttLC1CCCttCtgaCCttvctgttg:04 3 aggtC'vagtCtdaCUgCtmwC2.g3221231aC213taCaaga.Ca21CtCCCCC2tgthtggaCagCganggagCttCtth x tcta‘thaaCtthtCtnthacaaCtC3.10zatagcaocaaUgaattcyttttttcctgctCagtaatgcatgaggCtctgca 5 3 " ID ) 3 Cgtathggtt'tnCC132232123{nggitCCCttdz- CCangtoaCtC ______________________________ huCD37—57 agC-ttCCCaCcatgggCtggwgC 31 :atcattCtgrttCtootgC acaccaaCthCttcaC233,1:0212TtCCaaCtj cagggagCLigCC-C‘Cfiga'tCC-toaadCcaCtCtCagtcaCta3gctgac2twt31CthCa'fCWCL21igcattacctc‘ aggcttc37:0ttggC2 ttvodtmeathCCCCgaaaACotCt;:10r,tggatgvggmCattCtgtaCdQngCagta 1 cagfitattCaCCCtCCtt:32:atCtdggatatCattCfiCaCCtCatacafi3.0213213at1WCEYLC‘C"‘§.,Ctga£ECi‘V" : gtcaCCCgccgcagac21C30CaaCCtattdttUthtwggflfiCtdCCCmatgfichatayttCOCCtdtttzwrszC1 3 gr:g23C2CLCCYLUaCCthuchchCCCC2132123,;ggCCC231Ca33ttttCCCCtthCtCCmf‘ttctuaatCCdeg ngtggaaCarrCt'iCdCtgggatCCCthttaaaoattatttCCCtUagCCCgtCaCaCt”Ct'roaatagcggagCat tgacttmggtgt3T"23C23CttttCCC3rC{Giotto&OtkCECCQgTLfgtaCTKaLTgtC2tgtfitCm3123CCrrtwcttCta toomaCCC2131.1chatCtUtaaCCtCaaC312323233.CatCCaaCaCaaaqgtggatadzaaftgttgaaCC 2.2311621:th£2,1magacac21tacatguCCtCCttUtCCthCthamCCtCCtcggaggtCcatCtgtgtthtgtttCCC CCC323aCCCa'tgfiCthCtCttawatCtCthtaCa0athC21CC.tgCattwttgtCtt'tcvtgagC23tg2331g23tCCC gaggttaaattCaathtngggdtggwtm UgttcaCaatgccaagaCCaaOCCCdgg‘21g31agC31-21t2tt2321tt ctaCi-tta‘tcgggt21;;thCgt:gttCtCachtCC‘t313Cdegdfi“‘JLiCdcflggddégigdgtfiCaaott?0&§lggt9ffijc aacaaggctcttccCchCitCCCttmaaaaactatCtCcaaaUCCaaggcagCCanwaaCCC‘aggtetatacat EQCCCCcatCtagggacga C ggggttttaCCcttCtg‘2332231131; ‘ ' ‘ ‘ ‘ ' “ “ “ 3323-3 ' ' <‘“3223C63.923gc 4 4 \\L“..........,,_.w~aaaL““A. Caiaatgcafgawc"LLLLLLLLLLL‘‘L w________ L“\“FL‘LLLWLLLLW” Antlbod E 3 Od&EECELLfiLCLHé3thLLoaLiLagtLLtgggLfigctotthgIOthtaC'xgatg"*agaigtLaLa‘iLLag aigaLtLagiLmLagL.CECLL‘LE’ELtgiaioigtgggagaaadgtcaooatcaoatgtLgagoaagigagaaLaitLgCa gtaatttag0atggtatoagcagaaamgggaaaatotoCtCagCEcctggmaaigflgcaacaaaofiagoagatggtgi gCLaLoaagCfiLagtvgcagtfi‘O‘atcaggcaoaoagtafiocottcaagatcaaoagcctgcagtczgaagatmggga CfiattactgtoaacattaitggggtaotacgiggaogficthgaggoaccaagotggaaatcaaaogtacQfitggow cazccatLtgtofiLatLt o{LwaigaocaOECaaatLtggaaLfgLoiatgitgwigCotontoaamaLfiota tCLCagagaggoLaaagtaCaoioLaaggtogataachLoC’iLoaatgggiaaotoccaggagagtgto3Cagagc arigéLaocaaggaagoaoctaLCOLL?‘agoagoaocotgacgotgam433LgLagdLiaC"agdaawcaaaot oiaC0LLthoaaotLaLoLanagggoctgagotogcocgtoaoaaagagcitLaacaggggagagtgfiag3 v“n“... .LLLLLLLLLLLLLL“ “““LLLL‘LLL—Mg“4.(son)3359.162) mhuCD37-3 aa’tLgCLaLLatgth . LgLaLLaLttott:CtgigchaoagocaccggiLtiLauLigatamLaaatoaL (1.0 and 1.1) i ECLiachLttCLagtttgtaagigtjggigmgcgtaacaatoaoLtomgdLoiagiaaaacaiccgoagta 231i.thLaiggtacoaaCaaaagoLaggiaavts:aLLtaaacthtc:ItomtwfigotuowCLtcvotgatvgtUtgo oiioaogatiLiLiggficaggttongLoamfioatiadgaicaaLtLaLtLoaaCLaCaagaitingiacama LtgtoficatomC4ogcoatotgatgigcagttgaaatctggaaotgogc:“LCgtgLLLgLigaaiaaLttCtaICLod gagaggoCaaavtaoagtggqawtggataaogcoctcoaatcgggtaaLE'LCLL4aggg.smgtglLaciigagoaggao L acamaLLtaoaOCLtLagCgem:oCtgachtgagcaaagcaqaL wgagaaaczxsaaagtotacgc L K : 3 otfiomagime canggLotGaCLtgoC-CgiLacaadgaCLtiLaai'gggagagtgttag (SEQ ID C NO: 163) \ chCD3712 \ gamogLCaCLai0ggflggtofigtataatCLtgttLtivvEggLL4chLgLmagchmgataotgutafigiactoact \ “Mann“...m... \ CGgtoaooagcoagtCtggCagiothLtggPocagcgtgocaocaCicctgcogggoLtoaLjttoCGIgaooaCta 3 fiootatciotaotggtfio:aLagaeLocCaggasacoCLoiaLg:gtCigatcaagt'(LochtccaaCCrcgo \ CagoggcortwoootagafioLLiovtiLCoLLwCooaaotoamoao‘mgaaLa{LLaLLLLgitggggaagagga _.... taCLgCLaLfiaLLtaItgtLaacaotcfigggacattLLKaLaLLm0Gaagamaaoaaagotogaaatt'aagcgtzwg Wfluflfl.”flu—”y,"5...... gtggLWLdL:tctgtcfioatcttcocCLC:1thgatgaUCaatLgaaatN0gaaothCtotvfificivodgdgdai aacttomtcocagagaggLfaaagtaCagtgoa.aag.g0aiaaLCLLLiLLaatLLgUEaaL{L‘ooao0agagtha caoagoaggaLagCaagg:agCaLLtoLagLotoaocavmCoigac(5cigagLaz=a0Ca<>aLtacgavaaaLa L'aaagtotaorrLLtoLgaagtaCCLatcagggoCdgagotogcocgt'tascaaagzthtLaamggaqagangfia I Nmmmmw“ -~—»~»--—»« ~- —-— chCD37—3 8 LaLC'ttghLLiggtLLtg afLatCLLgtttLtLgtggoLaoamacéggtgficaitchagafimgdgdoof LaatLaLoaUCEafiatgtLogLagerCLCggCoagaaaatfiaLmioaatgtzagC2L1:gthctotgacfiaoaf ...... _;cattggtatoaaLagJAgLL,ggtaco:chC“aanogfiggaflmogaodwfiw ggLLthggaotLCLtCa4 ‘LCanggaootgnmooggoaLagtiatLaLtgchatatLCLCtaLfivm:igaagaUerLLaLmnta itgtcaaoaatggamotaaoufiCCocoaLCitggthngaaLadagLtgflagangogaLLUtfigLWCaLLaL L otgtottLElK/HLLLLLKatLtgp'Hg‘hOk/imga"(1E LaaLLGLLLLtottotthCtgotgaataaaofictatLLoa \ gagaggoasaviacafitgmawtggataaLgCLCCcoaa‘ngggiaCocaggagagtgtcawoaaoaqgao 3 .LgceLaggaL30LaCLtaLavLLtageagoaCCC.gacwctgagL(1:130agauaogagameaaagtctacgo otgLg3.agtLaLCCaimgggoowc,agotcgCLgodagaagathiLaacaggggagagtgtiag (SEQ T53 M3365) .34._V_______.,...........u..auu“an“............. huLDfl38 C70tLogLLaoLaiOggatggtLLtgLafiafttgmflwtwocacgLCBCtggficaLLo‘macmotgtcaca .agtC‘LCLagLLLLaLtg[LigLi‘tCCLLoootfiagooggtvaLoatgaLgctotgCcagficotoogtgacatamtgo Ci‘LaaaaaoatggmotaCoaLaottcaaaaofigcat'avgogfioctocoa ‘ attogtatoagoaaaaaoccvgt'o 0215.333:"”5bgifliflgbtvtoobfiticscatacagtotngcattagtELLatogaacdggaga’rgcagccaoCmLiaCtgt aaLoaaaowvavaiaaam0&3.ngggLot-cacoa‘mfsw ““17“.L“ .w m“L““LL‘L‘LLL‘LL.

W0 35740 PCT/U82012/031648 icttcatcttCCCOCcatCtgatgaoC.agtwaaatctggaaC.EgCCtCtJttgtgLCEgCEgaataaCCtC.taECCCagagaogccaaagtaCagtggaarrgttwatcmCg1.CCECcaachggtadCtCCCaggagagtomEEC-3oaacavgacaficaaggz(ECCagCaCCtaCCgCCtCagcagCaCCCtCaCCctfiagaaaocawCECECCCwaaaCaCaaaotCEaCCCthCgaagECaCCatCagggcctgagCtCUCCCgECaCaCCagagCthaaCanggdrragwttag (SEQ ED0 CCCCCCCCCCC CCCCCCCCCCCCCCCCCCC CCC__C._.....C CC............

CCC. CCCCCCCCCCCCCCCCCC “(11:thaCCatzqgttogtCatocatwtCtgttCCCCqer’CaC0C1ggagtauiawtwCECE’EO‘ECCELEEC CCaddgtCCthtdCtatgtCth.CagcCCCEggagdgCgtgtgamatgacttuELEgLECELCCaa‘VtgtgaCatacar qcattggtatCagcaaaagCCtggCCCa‘CCCCCtaaaaggtggatctacgataCttCtaatcthCatangtgEUCCC0C aaggttCtCCGggagtgg"agtvgcaCCCOfiatagtctC'tccatcavttcaatggaagcagaggatgcagcaaCCtdttC attotCauCagtogthgtttaatCCCCCtaCtttthICItggtaCaaag-tvgaoaEtaagcgtacge’tggctficacmt ctgtCEtCatctEC.CCUCCatCtgatgagcagttgaaatctooaactscCCctfittgtgtOCCEgCtgaataaCtECtatCCCC21 E35 (-£9 {:3O 0"? {'3C ('215-4» ('130 S12{21)W(38OCE‘TJfl3) 311)(D ,4.(3 (.3 (2 a: 0‘53 (3:; 9.2(1'2”‘3:'3».(—1- If;P4 ('2m f”) {3) (TC? ($2)? 1 3" "109:1:GM 52':Q {If}0 ('3 '99E"C.)ionIn,200 330HQa»).0‘"(IQ$1)(7%33H“E3321: ..... agCaaggaCagCaC.CtaCagCCtCCgCagCCECCCtg1.OCtoagCaaagCagaC‘CCECvagaaCCmagic50381894 Cg CthgaagtcaCCCCtCCEggchtgaqctchCCCCgtCCC2aangCttCaCCCgggaoagtgttavg‘EEQ EDC N0113]} huLD3/5E" gaa‘ttchC.caCatgggamgaCctgtattattCtottCtggtthtaCtht'ECtgCgt C<Y£Egdta0ECCECEC CcagaggcattggtaCCchCa‘CcatgagCCCCtCCcCEUngaGCgactgactatoaCttottCCUCCaCttCttcast.aCCtatat tcagcagamm1:0gaCCgtCtCCCCEa(*CgttgggtttacgacamtCCaaCCtggCttCCCCmCO’EtCCth taggttcagcggatC‘tgCgCEggcaoaagttamathaCCattwttCCatooagfiCCCaaoatocCgCtaCttaCtCEC E tgtcagcagtggagC23.CCECECCCCCCtaC'EttcggUCdgaCECtathogagatca‘EaCtacggtggC‘tgcaCCa .‘ tCtgtCttcatcttCCCgCLaECELaEOclO0124.055)afiaECEEQEdciW°CECEUEEgI0EgCCEOCIOEaEfi'ECE’ECEaECCCC. 1 gagaggCCaaaOE'tcaathCEamtogat'tachCCtCcaa’CnggtadCCCaggCad9EOEL8k8g&9939gdC 1 11:; E agC;saggaCagCaCctacachtCagcagcacmz gaCgCtCagC'EaagCagaCtEsmdaca‘aaagtctaCgC ' 1 CtgcgaagECdCCCatCaggCCCtoaUCECgCCgtcaCaa1gCgCtCCaacagg gagagtgttagE‘EEQ ED E N‘IXEESEE) huCD3756 gaattcgccCECCCCEgggctg CCtthEEWECCtgtttCtggtgCaathaCtgggfittca".C‘tgatatthCCCgaC acagagftCCdgCCEtCaEgagthItCtCchgaGaClaw0fECaanaCtEgttCagCt‘CCCCctcccgtcacatacatg cattggtaccagcagaagcctgaCaEgagthta13021CCtogatCtatUataCaagCaaECtggC'ttccggtgtCCCCEC ' aaitco 1Cate/t1110mmC.(31:1:aCC.'tEC:‘-‘C:CtCaat10211100322112,211:OCEOCEEECatatt 1 CC *CttttcaggcggcE: 150%) 27 Fara 07: I: 1 aCthCaecaatooatcaocCCCcCctCCtCCCEtCCCgCCCC~aaCaa'tattfig'tdattadetaC:r_,gtgg;g1‘:.1:game; atctgtCtthtCttCCOCCatctoatoagcagtt»aaatCtggEacthCCttCg‘towthCtgctaataacttCtatCCC4 agagaggCcaawtCiCaotggaaogtqqmfiadchcCtCCaatcg{gtaactcccaggagagtgtcacagagcagga CCaCaoC'tcCtCCCCUCCtCavcaocCaCCC‘mac‘rtgagccaaagcagactzngagaaaCacaaagtctacg CC‘thgaagtcaCCCdtCagggCCtfiaaCthCCCthacaaaUaaCttCadCaggggdgagtgttag (SEQ ID NO:E69) EEuCEB?57 = gaatthCCdccatggggtggtC*tgtttatCCtottcthtcgCaCCCCCCCCECaoocgtaCthgaOotcgtvttva aocmaCCcaccatgtcchttCCCCgoogaoaga*acaatUaCttOCCCgCCaCaCEOCCCzotaaCCtaC that11mtdCC91<>CaaaaacCag113301CCCgtcgttggatttaEgatCtCCtCtam.CEOOCEECagCgEtCCtg 1 1 CCCgctttt‘Ctttotaatgg'ttngggCttcctdEaOCCttaCCataaOCECCCthoaaUcCwaawCCgCtacataCta 1 ctgccagcagtggagtgataaCC.CCCCCCCCtthggCaggngCCaaat‘ggaoatcaaacgtangtggCthCCC 1 atctgtCtticatCE‘ECchCCCtCtgatgagcagttgaaatCtggaacEgCCtCtoEtOEgtoCctgctgaataacttctaCC 1310aGagchaaagtaCaCztgfiaaggtgataacocCctCC'thWstdaCtCCCaggagaotgtcaCCC-agt.gga cattcaaggacaUCdCCtaCCGCC[CagC'EgCa.CCtganCtgdt—CcaaetvcagactaCgagaaacacaaagtctacg g CCthgaaotCCathggg-CtgagctcgCCCgtcacaaagagc :1acaggggagagtgttag (SEQ EEE N0E201 513G225} Also CCE is a poEymacEeoEECEC having at ECast about 95%. at ECaSt about. 96%. at. 11:21:11; about 97%, at 583551: about 98%, m at ECast about 99% sequchC identity to SEQ ED NOS:E‘2E—E7€), E83. or E83.

Thus, in 12611211311 em‘bvdiments, the cECCtiCEC comprises (a) a. poEynuCECC‘CiCEC E1avi1‘1g at ECasE about 95/6 sequence EEEV t0 ‘1)“Q ED N05: 121‘ ~ E 35 E524. E, or 182., and/01” (E3) 21 pCEynuCECCtECEC having at 11321211: CEbCuEQ5/CSCC1CCCCC Edcnttty to SEQ ED NOS: E36 ESE E62 E76, 0': E8E. E11 Cartam embodnnents. the W0 2012/135740 .. 7g} - polynucieotide comprises (a) a polynucleotide having the nucieic acid sequence of SEQ II.) NQs: 121— 135, 152-161 or 132; and/or (b) a polynucleotide having the nucleic acid sequence of SFQ II.) NOS: 136»- 151,162—170, or .183.

In same embodiments, the pclyiiucleetide encodes the iight chain encoded by the recnmbinant plasmid DNA phuCD37—3LC (ATCC Deposit Designation 722, deposited with the ATCC on March 18, 2010) or a light chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% to the light chain encoded by phuCD37-3LC (PTA-10722). In some embodiments, the polynucleotide encodes the heavy chain encoded by the recombinant plasmid DNA phuCD37-3HCV.1.0 (ATCC Deposit Designation PTA-10723, deposited with the ATCC on March 18, 2010) or a heavy chain that is at least about 85%, at lesat about 90%, at least about 95%, or at least about 99% identical to the heavy chain encoded by phuCD37—3HCV.1.0 (PTA—10723). In certain embodiments the polynucleotide is the recombinant plasmid DNA phuCD37-3LC (PTA—10722) or the recombinant plasmid phuCD37- 3HCv.1.0 0723).

In certain embodiments the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g. a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

] In n embodiments the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the d polypeptide. For e, the marker sequence can be a hexa—histidine tag supplied by a pQE-9 vector to provide for ation of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a lutinin (HA) tag d from the za hemagglutinin protein when a mammalian host (e.g. COS-7 cells) is used.

The present ion further relates to ts of the hereinabove described polynucleotides encoding, for e, fragments, analogs, and derivatives.

The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the d polypeptide. In some embodiments, tide ts are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to a 7} _ optimize cedon expression for a particuiar host ifchange sedans in the human mRNA to those preferred by a bacterial host such as E.. call). {00231} Vectors and cells comprising the polynucleetides described herein are also provided.

IV. Metheds 9? use and plmmraeetxticai eompesitiens £00232] The CD37—binding agents (including antibodies, immunoconjugates, and polypeptides) of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment of cancer, such as B—cell malignancies, autoimmune diseases, and inflammatory diseases. In certain embodiments, the agents are useful for depleting B-cells. In certain embodiments, the agents are useful for ing autoreactive s. In certain embodiments, the agents are useful for depleting peripheral B-cells. In certain embodiments, the agents are useful for preventing inappropriate T—cell stimulation. The T-cell stimulation can be in connection with a B-cell pathway. The methods of use can be in vitro, ex vivo, or in vivo methods. In certain embodiments, the CD37-binding agent or antibody or immunoconjugate, or polypeptide is an antagonist of the human CD37 to which it binds.

In one , anti—CD37 antibodies and immunoconjugates of the ion are useful for detecting the presence of CD37 in a biological sample. The term "detecting" as used herein encompasses tative or qualitative detection. In certain embodiments, a biological sample ses a cell or tissue. In certain embodiments, such tissues include s that express CD37 at higher levels ve to other tissues, for example, s and/or B—cell associated tissues.

In one aspect, the invention provides a method of detecting the presence of CD37 in a biological sample. In certain embodiments, the method comprises contacting the biological sample with an anti-CD37 antibodyunder conditioes permissive for g of the anti-CD37 antibody to CD37, and detecting whether a complex is formed between the anti—CD37 antibody and CD3 7.

In one aspect, the invention provides a method of diagnosing a er associated with increased expression of CD37. In n embodiments, the method comprises contacting a test cell with an anti-CD37 antibody; determining the level of expression (either quantitatively or qualitatively) of CD37 by the test cell by detecting binding of the anti-CD37 antibody to CD37; and comparing the level of expression of CD37 by the test cell with the level of expression of CD37 by a control cell (e.g., a normal cell of the same tissue origin as the test cell or a cell that expresses CD37 at levels comparable to such a normal cell), n a higher level of sion of CD37 by the test cell as compared to the control cell tes the presence of a er associated with increased expression of CD37. In certain embodiments, the test cell is obtained from an dual suspected of having an autoimmune disorder or inflammatory disorder. In some embodiments, the disorder is associated with increased expression of CD37. In some embodiments, the disorder is associated with increased number of B—cells. in some embodiments, the disorder is associated with increased activity of B—cells- W0 2012/135740 PCT/U82012/031648 _ 72 _ In certain embodiments, a method of diagnosis or detection, such as those described above, comprises detecting binding of an anti—CD37 antibody to CD37 expressed on the surface of a cell or in a membrane preparation obtained from a cell expressing CD37 on its surface. In certain embodiments, the method comprises contacting a cell with an anti-CD37 antibody under conditions permissive for binding of the antiC‘D37 antibody to Cl3_,'I, and detecting whether a x is formed between the anti—CD37 antibody and CD37 on the oeli surface An iplaiy assay for detecting binding of an anti—C5937 antiboC'lV to C12733, exptessed on the suitace ot a oeoilis a "FACT 3" assay {68237} Certain other methods can he used to detect binding oi: antiC{”33.1 antibodies to CD37, Such s inclode, but are not limited to, antigen~binding assays that are weell known in the art, such as.< westein biots, radioimmunoassays, ELlSA (enzyme linked inimunosorbent assay), ”sandwich” imniunoassays, imniunopreoipitation , fluorescent innnnnoassays, protein A iminunoassays, and innnunohistooheniistry {lHC}. {£30238} in n embodiments, D37 dies are iabeied. Labels inciode, but are not limited to, iabels or moieties that are detected ly (such as fluorescent, ehromophoric, electron-dense, uminescent, and radioactive iabels), as well as moieties, such as enzymes or ligands, that are detected indirectly,eCg”tinonsh an enzymatic reaction or iar interaction } in certain embodiments, anti—CD37 antibodies are immobilized. on an insolnbie matrix. immobilization entails separating the anti-CD37 antibody from any CD37 that remains free in solution. this eonyentionaily is. accomplished by either insohihiiizins, the anti-Cl337 antibody hetero the assay procedure, as by adsorption to a watei’ninsoluhle matrix or surface (Bennich et al, US. Pat. No "3,72 ,7603, or by nt coupling (for example, using glutaraldehyde erossuiinhing), or by insolahilizing the anti~CIi33IantibodV after torination of a eoinpiex n the anti-CD3antibody and CD?"3 , , gby imntunoprecipitation.

{N32436: Any of the above embodiments of diagnosis or detection can be carried out using an oeoniugate ot the invention in place oi or in addition to an 337, dy. in certain embodiments, the disease treated with the C3D37~hinding agent is an mune or inflammatory disease. In certain embodiments, the autoimmune or inflammatory disease is selected from the group consisting of psoriasis, dermatitis, systemic scleroderrna and sis, responses associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult atory ss syndrome (ARDS), dermatitis, meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, leukocyte adhesion deficiency, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud’s syndrome, autoimmune thyroiditis, allergic encephalomyelitis, Sjorgen's syndrome, juvenile onset diabetes, immune mediated by cytokines and "iliyinphooytes, responses associated with acute and delayed hypersensitivity PCT/U82012/031648 _ 73 _ tuberculosis, sarcoidosis, poiymyositis, granulotnaiosis, itis, pernicious anemia (Addison’s disease), diseases involving leukocyte dispedesis eentt’ai nervous system (CNS) inflammatory disorder, multiple organ injury syndrome, hetnolytie anemia, myasthenia gravis, antigetnantihody compEex mediated diseases, anti~glomerular basement membrane disease, antiphosphoiipid syndrome, aiiergic neuritis, Graves disease, LambertEaton myasthenic syndrome, petnphigoid s petnphigus, autoimmune polyendocrinopathies, Reiter’s disease, stiff—man syndrome, Behcet e, giant cell arteritis, immune complex nephritis, lgA nephropsthy, igM polyneuropathies, idiopathic thromboeytopenic purpura (Ill?) and autoimmune throtnhocy‘topenia. {@242} in some embodiments, the autoimmune or inflammatory disease is seieeted from the group ting of: RA, inpus, immune thromhocytopenic purpurn, pure red ceiE aplasis, autoimmune anemia, coid agglutinin disease, type B syndrome of severe insulin resistance, mixed cryoglobuEinermis, henia gravis, r's granulomntosis, microscopic polyangiitis (MFA) refractory pemphigus vulget'is, omyositis, Sjogren’s syndrome, active type—ii mixed cryogiobuiinetnia, petnphigus Viligai‘is, autoimmune neuropatliy, paraneopiastic opsocionusarnyoclonus syndrome, and, relapsing remitting ntuitipies osi s (ERA/ESE {(33243} int:ertain embodiments the autoimmune e or inflammatory diseaseiscltarecterized by CD37 xptess1n- eelis to which the nding agent (eg.,antibody) binds, {.6244} Ehe present invention provides Eor methods of ng autoimmune and intiannnatory diseass tompiising istetingaatherapeutically effective amount of a CD3'7—bihding agent to a subject (tag, a suhject in need oE’treatment), in n embodiments, the subject is a human. {@245} The present invention further es methods for depleting B-cells, e,g., autorcuctive B» cells, described herein. En n embodiments, the method of .sittgth antibodies or other agent: ing B—ceiEs comprises contacting a B~ceil with a CD37~hinding agent. (eta, antibody) in vitro. For example, a eeEl Eine that expresses CD37 is cultured in medium to which is added the dy or other agent to deplete the cells. in some embodiments, the ceEEs are isoiated from a patient sanmie such as, for exatnpie, a tissue , pEeurai effusion, or blood sample and cultured in medium to which is added an CD3 7~hinding agent to e the. ceils. {@8246} in some etrthoditnents, the method of depleting B~eells, eg. autoretetive 3-ct.lls comprises contacting the cells with the C3E337~hinding agent (cg, antibody) in viva, in certain embodiments, contacting a red with 3 {i337binding agent is undertaken in an animal model For example, CD37- binding agents can be administered to xenografts expressing one or more CD375 that have been grown in immunocompromised mice (e.g. NOD/SCID mice). In some embodiments, cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and ed into immunocompromised mice that are then administered a CD37—binding agent to deplete B-cells. In some embodiments, the CD3 ing agent is administered at the same time or shortly after introduction of W0 35740 PCT/U82012/031648 _ 74 _ cells into the animal. In further examples, CD37 binding agents can be administered in vivo to mice expressing one or more CD37 antigens. In some embodiments, these mice can be engineered to express human CD37 in addition to, or instead of, murine CD37. In some embodiments, these mice are disease models, e.g. models for autoimmune disease. In some embodiments, administering a CD37 binding agent depletes B—cells in vivo. In some embodiments, a CD37 binding agent prevents T—cell ation. In some embodiments, administering a CD37 binding agent prevents or alleviates an autoimmune disease. [00247; in certain embodiments, the B~celis press CD37, in ether embodiments, the B-ceils do not overexpress CD37. In some embodiments, the B—cells are not cancer cells. in some embodiments, the IRS—cells are not turner ceiis In some embodiments, the B-cells are not cancerous cells.

] The t invention further es pharmaceutical compositions comprising one or more of the CD37-binding agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in treating autoimmune and inflammatory disease in human patients.

In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a pharmaceutically acceptable vehicle (e.g. r, excipient) (Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, ic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g. less than about 10 amino acid residues); proteins such as serum n, gelatin, or immunoglobulins; hydrophilic polymers such as nylpyrrolidone', amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; ydrates such as monosacchandes, disaccharides, glucose, e, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and non—ionic surfactants such as TWEEN or polyethylene glycol (PEG).

The ceutical itions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical (such as to mucous membranes ing vaginal and rectal ry) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, mal and transdermal); oral; or parenteral ing intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or ranial (e.g., intrathecal or entricular) administration, -75..

] An antibody or immunoconjugate of the invention can be combined in a ceutical combination ation, or dosing regimen as combination therapy, with a second compound having utoimmune or inflammatory ties. The second nd of the pharmaceutical combination formulation or dosing regimen can have complementary activities to inding agent of the ation such that they do not adversely affect each other. ceutical compositions comprising the CD37—binding agent and the second agent are also provided. For example, CD3 7-binding agents can be administered in ation with CD20-binding agents, such as Rituximab. In other embodiments, CD37—binding agents can be administered in combination with salicylate‘, nonsteroidal anti-inflammatory drugs such as indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g., ibuprofen and fenoprofen), naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, zomepirac and diflunisal; antimalarials such as chloroquine; gold salts; penicillamine; or immunosuppressive agents such as methotrexate or corticosteroids. In some embodiments, the CD37-binding agent is administered in combination with a second therapeutic selected from the group consisting of methotrexate, an anti-CD20 therapeutic, an anti—IL—6 receptor therapeutic, an anti—IL-12/23p40 therapeutic, a chemotherapeutic, an immunosuppressant, an anti-interferon beta-1a therapeutic, glatirareer e, an anti—a4-integrin therapeutic, fingolimod, an anti-BLys therapeutic, CTLA-Fc, or an anti-TNF eutic. In some embodiments, the CD37—binding agent is administered in combination with a second therapeutic that is an antibody directed against an antigen selected from a group consisting of CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD38, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138, and CD152. In some embodiments, the CD37—binding agent is stered in combination with a second thereapeutic that is an antibody directed against a target ed from the group consisting of IL-2, IL—6, IL-12, IL-23, IL-12/23 p40, IL-17, IFNy, TNFOL, IFNOL, IL—15, IL-21, IL—la, IL-lb, IL—18, IL—8, IL-4, GM—CSF, IL—3, and IL—5. In some embodiments, the CD3 7-binding agents are administered in combination with methotrexate.

For the treatment of the disease, the appropriate dosage of an dy or agent of the t invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, Whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient’s clinical history, and so on all at the discretion of the treating physician. The antibody or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is affected or a diminution of the disease state is ed. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody or agent.

The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. In certain embodiments, dosage is from 0.01 pg to 100 mg per kg of body weight, and can be given W0 2012/135740 PCT/U82012/031648 -76— once or more daily, weekly, monthiy or yearly, in certain embodiments, the antibody or ether CD3?— binding agent is given once every two weeks or once every three weeks. in certain embodiments, the dosage of the antibody or other CD3 ibinding agent is from about 0.1 mg tc abcnt 20 mg per kg of body weight. The treating physician can estimate repetiticn rates for dosing based on measured residence times and concentrations of the drag in bodily fluids or tissues. {9:325:23} The ation therapy can provide "synergy” and prove "synergistic”, ie. the effect achieved when the active ingredients used er is greater than the sum of the effects that results from using the compounds separately. A synergistic ef ect can be attained when the active ingredients are: (l) comforniulatcd and administered er deiivered aneousiy in a combined, unit desage formulation; (2') delivered by alternation or in parallei as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the cempcnnds are administered cr delivered sequentiaiiy, eug by different injections in separate syringes. in general, during ation therapy, an effective desage of each active ingredient is administered sequentially, i..e serially, whereas in combination therapy, effective s of two or more active ingredients are administered together, VI. Kits comprising inding agents The present invention provides kits that se the antibodies, immunoconjugates or other agents described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified dy against CD37 in one or more containers. In some ments, the kits contain all of the components ary and/or sufficient to perform a detection assay, including all controls, directions for performing , and any necessary software for analysis and presentation of results. A label or indicator describing, or a set of instructions for use of, kit components in a ligand detection method of the present invention, can also be included. The instructions f. One may be associated with a package insert and/or the packaging of the kit or the components skilled in the art will readily recognize that the disclosed antibodies, immunoconjugates or other agents of the t invention can be readily incorporated into one of the ished kit formats which are well known in the art. Such kits can also include, for example, other compounds and/or compositions, a device(s) for administering the compounds and/or compositions, and written instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products.

Further provided are kits comprising a CD37-binding agent (e.g., a CD37-binding antibody), as well as a second agent. In certain embodiments, the second agent is rituximab. In n ments, the second agent is methotrexate.

* * * Embodiments of the present disciosure can be further defined by reference to the following non-limiting es, which describe in detail preparation of certain antibodies of the present disclosure W0 2012/135740 _ 77 _ and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to als and methods, can be practiced without departing from the scope of the present disclosure.

It is understood that the examples and embodiments described herein are for z‘ative purposes only and that various modifications or s light thereof will be suggested to s skilled in the art and are to be included within the spirit and purview of this application.

All publications, patents, patent ations, internet sites, and accession numbers/database cited herein are hereby incorporated sequences (including both polynucleotide and polypeptide sequences) by reference in their entirety for all purposes to the same extent as if each individual publication, patent, and individually patent application, intemet site, or accession number/database sequence were specifically indicated to be so incorporated by reference.

Example 1 CDstXUesswnInnomalhumanJlBMCs The CD37 antigen was reported to be expressed on B-cells from the pre—B stage to the peripheral mature B—cell stage, while being absent on B-cell progenitors and terminally differentiated plasma cells. (Link et al., 1987, J Pathol. -21). In addition, the CD37 antigen is only weakly expressed T—cells, myeloid cells and ocytes (Schwartz-Albiez et al. 1988, J. on Immunol, 140(3)905—914).

The ability of antibodies (including certain CD37 antibodies and immunoconguates previously described in US. Published Application No. 256153, which is herein incorporated by reference in its entirety) to bind to normal human B—cells was measured using flow cytometry assays with fluorescently labeled antibodies. In on, the cially available QuantiBRITE system from BD Biosciences was used to estimate antigen density based on the number of antibodies bound to the cells (ABC). The QuantiBRITE system from BD Biosciences utilizes the following reagents: anti-CDZO-PE supplied at 100 ug/mL and QuantiBRITE PE ed as lyophilized PE-labeled beads. In addition, the huCD37-3 antibody was labeled with PE to obtain an dy—PE conjugate with an AbzPE ratio of approximately lzl.

Fresh buffy coats from healthy donors were obtained from Research Blood ents (Brighton, MA, US) as a source of normal blood cells. Buffy coats were prepared by centrifugation of a unit of whole blood and collecting the interface between the plasma and the red blood cells. This unpurified buffy coat contains PBMCs, neutrophils, platelets, red blood cells, and plasma and was used _ 73 _ for experiments on the same day it was drawn. Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats by standard density gradient centrifugation using Ficoll-Paque as follows.

Blood was diluted 1:3 with 1x HBSS containing 5mM EDTA and up to 30 mL were added to a 50 mL conical tube. Ten mL of Ficoll-Paque (GE Healthcare) were slowly added to the bottom of each tube.

Samples were centrifuged at 500 x g with no brake at RT for 30 minutes to obtain a layer of PBMCs below the plasma and to remove red blood cells and most granulocytes. The PBMCs were erred to new tubes and washed twice with 1x I-[BSS containing 5mM EDTA by centrifugation at 400 x g for minutes at RT. Staining buffer (1x HBSS, 1% BSA, 0.1% sodium azide) was then used to resuspend the PBMC pellets at 6.25 x 106 cells/mL. Eighty uL of cells were erred to a round-bottom 96-well plate to achieve 5 x 105 assay and 20 uL of human serum (Sigma H4522) were added to block Fc or- mediated binding and incubated with cells on ice for 20 min in the dark. Fluorescently labeled antibodies obtained from Miltenyi were used to identify PBMC populations: anti-CD3-allophycocyanin (APC) was used to identify T-cells, anti-CDl9-APC for B—cells, anti—CD56-APC for natural killer (NK) cells and anti-CD14—APC for monocytes.

Cells were co-stained for CD37 expression using 20 uL of huCD37—3-PE for a final concentration of approximately 10 ug/mL. Likewise, cells were co-stained for CD20 expression using 20 “L of anti-CD20—PE. As a control a non-binding PE-labeled hngGl e control antibody was used at ug/mL. Staining was carried out for 1 hour on ice in the dark. Samples were washed twice with staining buffer and fixed in 200 uL of 1% dehyde in 1x PBS. s were stored at 4°C in the dark until acquisition, which was performed within 4 days of sample preparation.

A fresh tube of QuantiBRITE beads was reconstituted in the supplied tube with 0.5 mL of staining buffer just prior to sample acquisition. Samples were acquired on a FACSCalibur flow cytometer (BD Biosciences). Compensation ls were run with each assay to select riate instrument settings and at least 10,000 events were collected for each sample. Instrument settings for fluorescence and compensation were kept the same for both cell sample and bead sample acquisition to allow for an accurate comparison. CellQuest (version 5.2.1, BD Biosciences) was used for acquisition control and analysis.

The QuantiBRITE is utilizes on a bead rd with 4 bead populations conjugated with a known number of PE molecules. For data analysis, a G1 gate was drawn around the bead singlets on an SSC—H scatter plot. This gated bead population was subsequently analyzed using a histogram plot of FL2-H to te the level of PE staining. Separate s were drawn around the peaks of the four bead populations (Ml—M4) and the geometric mean for FL2 of each bead population was determined. The FL2 geometric mean of each bead was plotted against the lot specific PE/bead values in a log—log plot. Linear regression was performed to obtain a standard curve using the following equation: y = mx + c, with “m” equal to the slope and “0” equal to the y-intercept. , 79 - {00265} For PBMC sample analysis, a GI gate was drawn around the positive fluorescent cell population of interest on an SSQH/TL-él-ll dot plot. This gated eel! population was subsequently analyzed using a histogram plot of FL2~H to evaluate the level of Pli~laheled antibody staining. The FLQ geometric well as mean was determined for each blood ceil sample stained with anti—CDB7-PE or anti—CD20~PE, as unstained control s. All geometric mean values for 3912 were d t the bead rd curve and values for PE per cell were extrapolated. Since both antibodyl’lfi a‘tes were at a PEzAb ratio of approximately 1:1, the values for PE per cell correspond to the number of antibodies bound per cell (ABC) value. Experiments were petitioned with duplicate samples for each assay. The mean and standard deviation was determined from several assays for each blood cell population CD37 expression was evaluated in normal blood cells from 4 independent donors. Results were compared to CD20 staining, unstained cells and a non-binding hngG—PE conjugate as ls. e of a typical staining profile of normal B-cells is given in a histograms in Figure l. The average ABC values of 4 ent experiments for CD37 and CD20 were calculated and listed in Table l.

Table 1: ABC values for CD37 and CD20 expression on human PBMC s N0 Ab hung-PE CD37 ABC {1320 ABC control control CDl9+Bcells 7744094598 “80 76 CD3+ T cells 2,016 336 74 68 CD56+ NK cells 3,090 264 85 88 CD 1 4+ monocytes 5,244 794 180 215 ] The highest overall CD37 staining level was found in CDl9+ B—cells at approximately 77,000 ABC. In addition, CD37 staining was seen at low levels in other PBMC populations examined, with CDl4+ monocytes showing CD37 staining at imately 5,000 ABC, CD56+ NK cells at 3,000 ABC, and CD3+ T cells at 2,000 ABC. Staining with the non—binding hngG-PE l resulted in ABC values of approximately 70 — 90 for B, T and NK cells and approximately 200 for monocytes. In the same 4 donors CD20 expression was evaluated in comparison to CD37. In accordance with published findings, the CD20 staining was restricted mainly to CDl9+ B-cells with an ABC value of approximately 95,000 ABC. The CD20 expression level was just slightly higher than the CD37 expression level. Only minimal CD20 staining was observed in other PBMC populations examined, with CDl4+ monocytes showing CD20 staining at 794 ABC, CD56+ NK cells at 264 ABC and CD3+ T cells at 336 ABC.

This result demonstrates that high CD37 sion is mainly restricted to B~cells in peripheral blood samples with only minor expression on peripheral T cells, NK cells and monocytes. This is consistent with published findings ((Moore et al. 1986, i lmmnnol. l3‘7(9):30l3—8; Sehwartnhlhiez et al, @833, J. hnnmnol., l40l’3)905~9l4). in addition, we found that the (2937 expression levels on PCT/U82012/031648 - 80 _ peripheral B-cells is similar to the level of CD20 sion. This expression pattern strongly t that CD37 directed therapies may be a suitable for targeting B—cells in diseases such as B-cell malignancies, autoimmune diseases, inflammatory diseases or other disorders of the immune system analogous to the use of CD20 directed therapies.

Example 2A InvztroBgduleelenonusmreurlfiedl’BMCs {99279} The ability of humanized antibodies to deplete B—eells was measured using in vine assays with human PBMCS according to published studies performed with rlmxiinab {'V'ugmeyster et al.

Cytonietry A. 2003;52{2):ll}l~9 and Vuglneyster et al. int phannaeol. 2004;4(8):l l l7—i24}.

Alernlnznmab (Campatli) was used as appositive control, since it has been reported to efficiently deplete cytes in viva and in vim: (Hale, Blood. 1933 Oet;62(4)2873—82 and Waldmann, Fllllos Trans R Soc Lond B Biol Sci. 2005 Sep 29;360(lZl-6l):l707-l E).

] Fresh buffy coats from healthy donors were obtained from Research Blood Components (Brighton, MA, US) as a source of normal blood cells for all experiments within this study. Buffy coats were prepared by centrifugation of a unit of whole blood and collecting the interface between the plasma and the red blood cells. This unpurified buffy coat contains PBMCs, neutrophils, platelets, red blood cells, and plasma and was used for experiments on the same day it was drawn. Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats by standard density gradient centrifugation using Ficoll-Paque as follows. Blood was diluted 1:3 with 1x HBSS containing SmM EDTA and up to mL were added to a 50 mL conical tube. Ten mL of Ficoll—Paque (GE Healthcare) were slowly added to the bottom of each tube. s were centrifiaged at 500 x g with no brake at RT for 30 minutes to obtain a layer of PBMCs below the plasma and to remove red blood cells and most granulocytes. The PBMCs were transferred to new tubes and washed twice with 1x HBSS containing SmM EDTA by centrifugation at 400 x g for 10 minutes at RT. Staining buffer (1x HBSS, 1% BSA, 0.1% sodium azide) was then used to resuspend the PBMC pellets in the initial blood volume to e the original cell density.

Z To assess: the effect 37-3. '?~3~SMCC£§M l huCD37—50~Si\v’lCC— , lnlCD37~50, DMl, mab, alemtnznrna‘n (Carnpath), and “l‘RtHllé on PBMC depletion. 90 at of purified cells. were added to l2 x 75 min polystyrene tubes and incubated with 10 tlL ofa 100 rig/mi, solution of each sample or a ltnlgG lsotyne control antibody for l ltr at 37°C in a humidified 5% (302 incubator. The final antibody (Ab) tration was ill gig/ml, in a final volume of 100 llL in staining butler. Three independent samples were prepared for each treatment. {9&273} To identify populations of PBMCS, all samples were co~stalned immediately alter Ab tion with l0~20 llL of lluorescently labeled Abs obtained from, for example. Bl) Biosciences or W0 2012/135740 PCT/U82012/031648 _ 81 _ yi. Anti-CD3-PerCP—Cy5.5 was used to identify T cells, anti-CDl9—APC for B—cells, and anti- CDl4—FITC for monocytes. ng was carried out in a total of 150 uL for 30 min in the dark at RT.

CountBright Absolute ng Beads (Invitrogen) were vortexed and added to each sample at 50 uL per tube. For PBMC prep samples, cells were washed once with 1 mL staining buffer and centrifuged at 400 x g for 3-5 min. Supernatant was removed with a 1 mL pipette and cells were resuspended in 500 [AL of 1% formaldehyde in 1x PBS. Samples were stored at 4°C in the dark until acquisition, which was performed within 4 days of sample ation.

TreeStar FlowJo software (version PC 7.5) was used for data analysis. A gate was drawn around the CountBright bead population on an FSC-H vs SSC—H dot plot to determine a total bead count for the sample. To determine the total count for each PBMC population of interest, a te gate was drawn around the positive fluorescent population on an SSC-H vs FL(x)—H dot plot, where x is the l of interest. Specifically, a total count for T cells in a sample was found by gating the positive population on an SSC—H vs FL3-H dot plot; for B-cells, the positive population was found on an SSC-H vs FL4-H dot plot; for NK cells, an SSC-H vs FL2-H dot plot was used; for monocytes, an SSC-H vs FLl-H dot plot was used. The ratio of CD19+ cells for B—cells (CD3+ cells for T cells, CD56+ cells for NK cells, or CDl4+ cells for monocytes) ve to beads was determined and multiplied by 100. Percent depletion was then calculated by taking the ratio of the cell to bead ratio in treated samples ve to the cell to bead ratio in isotype control treated samples, subtracting this from 1 and multiplying by 100. This corresponds to the following formula: Percent ion = 100 x (1 -cell to bead ratio of d sample/ cell to bead ratio of control sample). Data for all cell types was analyzed in the same manner.

For two donors tested, treatment of purified PBMC samples with huCD37-3, huCD37-3— SMCC—DMl, huCD37-50 or huCD37-50—SMCC-DM1 resulted in approximately 55-70% depletion of B— cells (see Figure 2). There was less than 10% depletion of T cells or monocytes. The B—cell restricted depletion effect indicates that this activity is linked to the high CD37 expression on B-cells. In comparison, treatment with the anti-CD20 antibody rituximab resulted in approximately 30—40% depletion of B-cells. Treatment with the anti-CD37 SMIPTM TRU-016 resulted in only 20—30% depletion of B—cells.

Alemtuzumab treatment ed in depletion of 60—70% of B—cells, 55-65% of T cells and 40-65% of monocytes.

Example 2B Dpseressonseformvztro Brcell d6551€41.91!.HSiIlE.§Z§11£if1§§iRBMQ§ ] To evaluate the dose-response of the antibodies and conjugates, purified PBMCs from 2 donors were incubated with a 5—fold sample dilution series. Each sample dilution was added at 10 uL per tube to 90 uL of purified cells in triplicate and ted for 1 hour at 37°C in a humidified 5% C02 incubator. The final tration ranged from 10 ug/mL to 0.13 ng/mL. The same amount of a non— binding hngG Ab was used as an isotype control. 2012/031648 _ 82 _ For two donors tested, treatment of purified PBMC samples with huCD37SMCC-DM1 resulted in a clear dose-response for the B—cell depletion activity (see Figure 3A and B). Incubation with huCD37-3—SMCC-DM1 caused in Vitro depletion of approximately 60% of B—cells with an ECSO of 40-75 ng/mL. For an additional donor tested, treatment of purified PBMC samples with huCDE7-3, huCD37-38, huCD37-50, and huCD37-56 antibodies also resulted in a clear dose—response for the B-cell depletion activity (see Figure 3C). Incubation with these antibodies caused in Vitro depletion of approximately 60- 70% of B—cells with an EC50 of 20-30 ng/mL.

Example 2C InvztroBcelld‘afletlonusmxwmlgblggd The y of zed antibodies to deplete B—cells was measured using in Vitro assays with whole blood according to published s performed with rituximab (Vugmeyster et al. try A. 2003;52(2)2101-9 and Vugmeyster et al. Int Immunopharmacol. 2004;4(8):l 1 .

Fresh buffs-'7 coats from healthy donors were obtained from Research Blood Components (Brighton, MA, US) as a source of normal blood cells for all experiments within this study. To assess the effect of -3, huCD37SMCC—DM1, rituximab, alemtuzumab (Campath), and TRU-Ol6 on peripheral blood cells (PBCs) in a whole blood matrix, 90 pL of whole blood from a buffy coat were incubated with Abs or isotype control as detailed above in a total volume of 100 pL. Three independent samples were prepared for each Ab treatment.

To identify populations of blood cells, all samples were co-stained immediately after Ab incubation with 10 - 20 pL of fluorescently labeled Abs obtained from, for example, BD Biosciences or Miltenyi. Anti-CD3-PerCP-Cy5.5 was used to fy T cells, anti-CDl9-APC for B-cells, anti-CD56— PE for NK cells, and anti-CDl4-FITC for monocytes. ng was carried out in a total of 150 [1L for 30 min in the dark at RT. CountBright Absolute Counting Beads (Invitrogen #C36950) werepvortexed and added to each sample at 50 pL per tube to allow standardization of cell counts.

Following cell staining, 2 mL of BD FACS Lysing Solution (BD Biosciences, diluted 1:10 in dH20 according to the manufacturer’s instructions) were added to each sample in order to lyse the RBCs t. Samples were incubated at RT for 15—20 min in the dark, centrifuged at 400 X g for 3—5 min, and resuspended in 500 [1L of 1% formaldehyde in lx PBS. Samples were stored at 4°C in the dark until ition, which was performed within 4 days of sample ation. Samples were acquired on a BD FACSCalibur. Compensation controls were run with each assay to m instrument settings. A total of 160,000 ungated events were acquired for each sample using BD CellQuest software (version 5.2).

TreeStar FlowJo re (version PC 7.5) was used for data analysis as described above.

For one donors tested, treatment of purified PBMC samples with huCD37-3, huCD37 SMCC—DMl, huCD37-50 or huCD37—50—SMCC-DM1 resulted in approximately 40% depletion of B— PCT/U82012/031648 _ 83 _ cells (see Figure 4). There was less than 10% depletion of T cells, 'NK cells or monocytes. As seen for puréfied PBMCs, the in vitro depletion is restricted to s indicating that the activity is linked to the high CD37 expression on B-cells. In comparison, treatment with the anti—CD20 dy rituximab or the anti-CD37 SMIPTM TRU—016 resulted in a less than 10% ion of B-cells. Alemtuzumab treatment ed in depletion of 40% of B-cells, 80% of T cells, 15% ofNK cells and 20% of monocytes.

Example 2D DovLQPOUNOEHVJWBLfilliltfiWflé—Lhfillfilfi..lllgfilél 3082283} To evaluate the dose—respense of the antibodies and conjugates? whole bleed from 2 dencrs was incubated with a lG—t‘eld sample dilution series. Each sample dilution was added at it? at. per tube to 90 til, 0f purified cells in triplicate and incubated for '1 hr at 37°C in a humidified 5% C703 incubator. The final concentration ranged from 10 ngij t0 ill ngx’rnL The same amount cf 3 nen~hinding hulgG Ab was used as an isotype central. 300284} Fer two sinners tested, ent of whole bleed samples with huCD37-3 or huCl'RS7—3— SMCGDMI ed in a clear dese response for the B~cell depletion activity {see Figure 5A and B). ln additlen, huCDBZLSlB was tested fer one (inner and also showed a similar dose response fer the B—cell depletion activity (see Figure SB}. lncnbatioh with huCl‘Bil—E, buCD373~SMCC~DMl er huCD37-v50 caused a maximum response at in vitro depletion 0f appreximately 304595 of B~cells with an ECSG of 40420 ng/mL.

In addition to the in vitro experiment described above, the capacity of CD37 antibodies to deplete B cells in vivo can be tested in huCD37 expressing mice (described in Example 3) and, for antibodies that crossreact with macaque CD37, in monkey.

Example 2E .m,Mm93;itchiinstsl..e«ase studlesusmwhumanPBMCs In vitro ne e was measured by ELISpot for IFN-y (Interferon), TNF—a (Tumor Necrosis Factor) and lL-6 leukin-6) using peripheral blood mononuclear cells ) from healthy human donors incubated for 18-20 hours with compounds at a concentration of 2.5 ng/mL to 250 pg/mL. The ELISpot method is designed to measure the number of cells secreting cytokine by capturing the cytokine onto the assay plate during the entire length of the tion. In all assays the positive control D3 antibody CD3-2, as well as a negative non—binding isotype hngG control dy was included. Alemtuzumab (Campath®) and rituximab (Rituxan®) were used in ison, since both have been reported to induce cytokine release in patients (Wing. J Clin Invest. 98:2819 (1996) and Winkler, Blood 94:2217—2224 (1999)). The assay ions were chosen to reflect conditions that are relevant for antibody therapeutics. The highest concentration of 250 pg/mL tested corresponds to the maximum W0 2012/135740 2012/031648 _ 84 _ serum concentration of an antibody, such as for example the CDZO-directed rituximab, in patient plasma after an infusion of 10 mg/kg of antibody.

As can be seen in Figures 6 and 7, the positive control anti—CD3 antibody induced release of very high levels of IFN—y, TNF-a and IL-6 with PBMCs from two different . In the same assays, alemtuzumab caused intermediate cytokine release, while rituximab caused moderate cytokine release with PBMCs from two different donors. In contrast, huCD3 7-3, huCD37—50, huCD37SMCC-DM1 or huCD37SMCC—DM1 did not cause significant cytokine release in our assays.

This underscores the utility of the described CD37-targeting antibodies or conjugates as therapeutics as they combine potent activity, such as B-cell depletion, with a ble safety profile with t to cytokine release.

Example 3 1.11_yi129._m9d9.1§.19.§xa1natit11§agixi§:.913.9123]..di:§§t§d_antihgdi§§_9r.,aniuaates B—cell depletion is known to ameliorate autoimmune es. In fact, rituximab has been approved for rheumatoid arthritis treatment (Edwards JC et al. Nat Rev Immunol. 6: 119 (2006)). In animal models, B—cell depletion using antibodies against B—cell antigens such as CD20, CD19 and CD79 has been shown to inhibit or ameliorate l autoimmune diseases including systemic lupus erythematosus (SLE), experimental autoimmune encephalomyelitis (EAE; mouse model of multiple sclerosis), type-l diabetes (TlD) and toid arthritis (RA). The CD37 antigen is expressed at high levels in human s. Therefore, antibodies or immunoconjugates directed against the CD37 antigen could potentially deplete B-cells and be therefore useful to treat multiple autoimmune diseases.

To test the utility of CD37 ing antibodies and immunoconjugates to treat human autoimmune diseases, the activity of such CD37 targeting antibodies and immunoconjugates can be d in mice using several murine autoimmune disease models.

For e, anti-murine CD37 antibodies can be generated using CD37-knock-out mice or other species such as rat and r, and antibodies that deplete B-cell in viva effectively can be selected.

The therapeutic ial of anti-CD37 antibodies can be tested in mouse models representing human autoimmune diseases, for example, a neous TlD model in NOD mice, a myelin endrocyte glycoprotein (MOG) peptide induced EAE model in wild type C57/Bl6 mice, a collagen induced rheumatoid arthritis model in DBA/l mice or a spontaneous systemic lupus erythematosus (SLE) model in MRL/lpr mice. Examples of murine CD37 antibodies and their therapeutic efficacy in s animal models of autoimmune disease are provided below.

] Alternatively, the therapeutic potential of anti-human CD37 antibodies and immunoconjugates can also be tested in murine autoimmune disease models that have been engineered to W0 2012/135740 PCT/U82012/031648 _ 85 _ express the human CD37 antigen. Such human CD37 (huCD37) expressing mice can be generated using standard knock in (Kl) or enic (Tg) approaches. For example, to generate huCD37 KI mice, human CD37 cDNA can be ed into the murine CD37 locus in the C57/Bl6 embryonic stem (ES) cells. The homozygous huCD37 KI mice will express human CD37 cDNA under the regulation of the endogenous murine CD37 promoter, thus the expression pattern of the huCD37 would mimic that of the endogenous muCD37. The different approach utilizes bacterial artificial chromosome (BAC) containing the human CD37 gene that can be ly inserted into the mouse genome. This transgenic approach has been used successfully to generate huCDZO Tg mice resulting in B-cell specific high level expression of the antigen. {(302193} The resulting huCD37 expressing mice based on the C57/Bl6 background can be used to further develop several autoimmune disease model. For examples, MOG peptide immunization in the C57/Bl6 strain background can s severe EAE in two weeks. In on, introducing a FcyRIIB knock out phenotype by ng huCD37 expressing mice with 6 FcyRIIB knock out mice should yield a mouse model that spontaneously develop SLE and develop RA upon immunization with collagen II antigen. atively, backcrossing of the huCD37 expressing C57/Bl6 mice into the NOE?) or MRL/lpr background for 10 generations can provide spontaneous TlD and SLE models, respectively.

Example 4A generatigugfamtianggugllflmgnmlgnal.an.tih9si§:;c_k>ne_2_§_2_:3_ To develop proof of concept that CD37 targeting antibody and immunoconjugate can inhibit autoimmune disease, anti—murine CD37 (muCD37) monoclonal antibodies were generated by immunizing CD37-knock-out 6 mice with 300—19, a murine pre-B cell line that endogenously expresses the muCD37 antigen. The immunogen was injected aneously at the dose of 5x106 cells per mouse every 2 weeks for 5 times. Three days before being sacrificed for hybridoma generation, the immunized mice received intraperitoneal injection of another dose of antigen. The spleen cells were fused with murine myeloma P3X63Ag8.653 cells (P3 cells) (J. F. Kearney et al. 1979, JImmunol, 123: 1548-1550) at ratio of 1 P3 cells: 3 spleen cells according to standard procedure. The fused cells were cultured in 640 selection medium containing nthine-aminopterin—thymidine (HAT) (Sigma Aldrich) in % C02 incubator at 37°C until hybridoma clones were ready for antibody screening.

] Screening was done using flow tric binding assay with spleen cells from wild type mice and nockout mice. Tie spleen cells were counterstained with anti—CD45R (8220) antibody to identify B cells that constitutively express CD37 n. The hybridomas producing antibody that bound the wild type, but not CD37-knock-out, B cells were subcloned by limiting dilution. One stable subclone (clone 252-3) was obtained. The 252—3 hybridoma was expanded in low IgG serum containing media and the antibody was purified using standard methods with protein A/G chromatography.

PCT/U82012/031648 -86— Example 4B The purified 252-3 monoclonal antibody was identified as a mouse IgG2a with lsoStrip mouse onal antibody irig kit (Roche Diagnostics Corporation, Indianapolis, IN). To determine the binding affinity to the muCD37 antigen, various concentrations of 252-3 antibody were incubated with 300-19 cells, a murine pre-B cell line that expresses the muCD37 n, for 30 minutes at 4°C. Cells were then washed and counterstained with anti mulgG—PE conjugate (Jackson Immunoresearch, West Grove, PA) for 30 minutes at 4°C. The cells were finally washed, fixed in in and analyzed by flow try using a FACSarray (BD Bioscience, San Jose, CA). The flow try data were ed using FlowJo (Tree Star Inc., Ashland, OR) and the geometric mean fluorescence intensity was plotted against the antibody concentration in a semi-log plot (Figure 8). A dose-response curve was generated by non-linear regression and the ECSO value of the curve, which coréesponds to the apparent dissociation constant (Kd) of the antibody, was calculated using GraphPad Prism (GraphPad Software Inc., La Jolla, CA). It was found that the Kd of the 252—3 antibody was 14 nM.

In contrast, the 252-3 antibody did not bind to human tumor cells expressing the human CD37 antigen.

The 252-3 antibody was then used as a surrogate antibody in murine autoimmune disease models to demonstrate the therapeutic potential of a CD37—targeting antibody for the treatment of mune diseases (Examples 5-7).

Example 5 i ..»..<.‘....._ Experémental autoimmune encephalomyelitis (EAE) is an animal model of inflammatory demyelinating disease of the central nervous system (CNS), including multiple sclerosis in human.

Murine EAE is commonly d by immunization of spinal cord homogenates, brain ts, or CNS protein such as myelin n or peptide, followed by injection of pertussis toxin to break down the blood-brain barrier and allow immune cells access to the CNS tissue. This immunization leads to multiple small disseminated s of demyelination in the brain and spinal cord, causing tail paralysis followed by limb paralysis.

To test the activity of uCD37 antibody in the EAE model, we first studied the capacity of the 252—3 antibody to deplete B cells in viva. C57Bl/6 mice were injected intraperitoneally with 25 mg/kg of 252—3 antibody or polyclonal muréne IgG (Jackson Immunoresearch, West Grove, PA) as a control. Peripheral blood was collected at different time points and analyzed for B and T cell levels by flow try, Allophycocyanin (APC)—conjugated anti—mouse CD45R (B220) antibody (ebioscience, WO 35740 - $7 ..

San Diego, CA) and cein isothiocyanate (FITC)—conjugated anti CD38 antibody (ebioscience, San Diego, CA) were used to stain B and T cell populations, respectively. B cell depletion was assessed by calculating the ratio of B to T cells for each sample and the B/T ratio was normalized by g the average B/T ratio of murine IgG—treated samples to 100%. The ized B/T cell ratio was plotted mngG control mice and 252-3 antibody treated mice (Figure 9A). The result show that the B cell level of the mice treated with 252-3 antibody was rapidly d within a few hours after the antibody ion.

The B cell depletion reached ~70% at 3h and peaked at day 3 (> 95%). After day 3, the B cell level slowly increased and reached ~60% of the normal level at day 14. This data suggests that the 252—3 antibody can rapidly and efficiently e peripheral blood B cells, and this effect was sustained for at least 7 days after the antibody injection.

The second study tested the capacity of 252-3 antibody to inhibit EAE. In this study, EAE was induced in C57Bl/6 mice by subcutaneous immunization of 55 peptide emulsified in complete Freund’s adjuvant (EAE kit from Hooke tories, Lawrence, MA) into the upper and lower back at day 0 and two intraperitoneal injections of pertussis toxin at 21": and 24h after antigen immunization. Mice were checked for EAE signs daily starting on day 7 after immunization. The disease severity was scored on a scale of 0 to 5 using the following criteria: Cl1nlcal Observations 0 ENO obvious changes1n motor functions of the mouse in comparison to non--immunized mice When picked up by the tail, the tail has tension and1s erect. Hind legs are usually spread apartE Whenthe mouse is walklng, there1s no gait or head t1lt1ng l Limp tail.

When the mouse is picked up by tail, instead of being erect, the whole tail drapes over your 3 finger. 2 Limp tail and weakness of hind legs.

When the mouse is picked up by tail, legs are not spread apart, but held closer together. When the mouse is observed when walking, it has clearly apparent wobbly walk. i E 3 Limp tail and complete paralysis of hind legs (most common) E Limb tail with paralysis of one front and one hind leg.

OR, ALL of: *8 Severe head tilting 8 Walking only along the edges of the cage a Pushing against the cage wall 1* Spinnmg when pickedupbythe tail PCT/U82012/031648 a 88 _ 4 Limp tail, complete hind leg and partial front leg paralysis.

Mouse is minimally moving around the cage but appears alert and g. Usually, euthanasia is recommended after the mouse scores level 4 for 2 days. When the mouse is euthanized because of severe paralysis, a score of 5 is entered for that mouse for the rest of the experiment.

“Massages,“_______.._...i.iiiiiiii..........v"" “bureau“......................................................................................................................

Complete hind and front leg paralysis, no movement around the cage. 3 OR, t Mouse is spontaneously rolling in the cage. I OR, : Mouse is found dead due to paralysis. i If mouse is alive, euthanize the mouse immediately if it scores 5. Once mouse scored 5, the same score is entered for all the days for the rest ofthe experiment. =iii‘dsiib‘‘1‘‘'1‘‘‘‘'"''Ali'iii{£5'é"é'i'e'fr'i'eifc'l'"iESEBB?‘v‘‘Ei;£,§ii“§ETf‘iiXiawwbethErT“13313“1‘3?£121};“é‘fiéFEiiiiii;EH"iiiiiiiii‘ri1221i}’cii’fK? the disease onset, mice were randomized and the 252-3 antibody or polyclonal mulgG was injected once intraperitoneally at a 25 mg/kg dose. A total of 10 mice were enrolled for each group. At the end of the study (18 days after the disease onset), the data were onized based on the day of disease onset for each mouse. The disease progression plot (Figure 9B) shows that mice from both groups had relapsing- remitting form of EAE. During the first wave of clinical symptoms, the control mice reached the mean of 3 while the mice treated with 252-3 antibody had a mean of 2. The difference in disease severity between these two groups was sustained for more than 2 weeks after the disease onset. Taken together, this data suggests that the 252-2 dy treatment rapidly depletes the B cell population and alleviates EAE.

Example 6 Anti-muQQ§.Z_m9n9919nraLanti.b9d§i__irthitzit§Eritreidiabe mlce Type-l diabetes (TlD) or juvenile diabetes or insulin-dependent diabetes millitus (IDDM) is caused by auto—immune reaction against insulin—producing pancreatic beta cells. Destruction of beta cells reduces insulin tion and increases glucose level that produces various al symptoms. TlD incidence in Northern Europe and the US is n 8 and 17/ 100,000. Insulin supplement is the most common treatment of the disease. ese diabetic (NOD) mice neously develop TlD and have been widely used to model the human disease. In NOD mice, the disease starts with leukocytic infiltration of the pancreatic islets (called insulitis) as early as 4 weeks of age. The tis progresses rapidly, leading to destruction of pancreatic islets and diabetes starting at 12-15 weeks of age. B cell depletion using anti-CD20 antibody in the early stage of insulitis has been reported to delay the disease onset (Hu et al., J Clin lnves. 117, 3857 (2007)), suggesting that B cells play a critical role in the disease pathogenesis in NOD mice.

W0 2012/135740 PCT/U82012/031648 To test the activity of anti-muCD37 antibody, the 252—3 antibody was injected into six female NOD mice intraperitoneally at 25 mg/kg every 10 days for a total of 4 injections starting at 5 weeks of age (n=6). The l mice (n=6) were injected with polyclonal murine IgG (Jackson Immunoresearch, West Grove, PA). Three days after the last injection, the B and T cell levels in peripheral blood were examined by flow cytometry. Allophycocyanin (APC)—conjugated anti—mouse CD45R (B220) antibody (ebioscience, San Diego, CA) and fluorescein isothiocyanate (FITC)-conjugated anti CD38 antibody (ebioscience, San Diego, CA) were used to stain B and T cell populations, respectively. The B/T cell ratio was ized to murine IgG control d samples as described above and the normalized B/T cell ratio was plotted for mngG l mice and 252-3 antibody treated mice (Figure 10A). The results show that the B cell level of the mice treated with 252-3 antibody was significantly reduced as compared to the control mice, suggesting that the 252-3 antibody efficiently depletes peripheral blood B cells in NOD mice. To examine the effect of B cell depletion by anti-muCD37 antibody, blood glucose level was measured weekly starting at 12 weeks of age. Mice with blood glucose level 2 250 mg/dL in two consecutive weeks are considered diabetic. The data in Figure 10B shows that the control mice started to develop diabetes on week 15 and 83% of the mice had diabetes on week 22. In contrast, the mice treated with 252-3 antibody started to develop es on week 17 and only 50% of the mice were diabetic on week 27. This data shows that treatment of 252—3 antibody efficiently depletes B cells in NOD mice, delays the onset of diabetes and significantly reduces the disease incidence.

Example 7 Collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) that is widely used to investigate e pathogenesis and to validate therapeutic targets. Arthritis is ly induced in mice or rats by immunization with gous or heterologous type II collagen in adjuvant.

This immunization elicits a robust T- and B- cell response to the antigen leading to proliferative synovitis with infiltration of rphonuclear and mononuclear cells, pannus formation, cartilage degradation, bone erosion and fibrosis.

Since different mouse strains have different susceptibility to antibody-mediated B cell depletion (Ahuja et al., J. l, 179: 3351-3361 (2007)), to test the activity of anti—muCD37 antibody in CIA model, we first studied the ty of the 252-3 antibody to e B cells in DBA/ 1 mice. Mice were injected intraperitoneally with 25 mg/kg of 252-3 antibody or polyclonal murine IgG (Jackson Immunoresearch, West Grove, PA) as l. Peripheral blood was collected at ent time points and ed for B and T cell levels by flow cytometry. Allophycocyanin (APC)—conjugated anti-mouse CD45R (B220) antibody (ebioscience, San Diego, CA) and fluorescein isothiocyanate (FITC)—conjugated anti CD38 antibody (ebioscience, San Diego, CA) were used to stain B and T cell populations, WO 35740 PCT/U82012/031648 _ 90 _ respectively. The normalized B/T cell ratio was was calculated as described above and compared between the mngG control mice and 252—3 antibody treated mice (Figure 11A). The result show that the 252—3 antibody significantly reduced the peripheral blood B cell level to ~20% and ~8% in l and 3 days after the antibody injection, and this low B cell level was maintained at 7 days after the dy injection. This data suggests that the 252-3 antibody can rapidly and efficiently deplete peripheral blood B cells, and this effect was sustained for at least 7 days after the antibody injection.

The second study tests the capacity of 252-3 antibody to inhibit CIA. In this study, CIA was induced in DBA/1 mice by subcutaneous zation of chicken collagen/CFA (complete Freund’s nt) on day 0 and chicken collagen/IFA (incomplete Freund’s adjuvant) on day 21 (Hooke Laboratories, Lawrence, MA). Mice were checked for CIA signs daily starting on day 21 after immunization. The CIA severity was scored on a scale of 0 to 16 (based on a score of 0 to 4 for each paw) using the following criteria: Clinics“, lons Normalpaw,“_ fiuOneto__e__ 1nflamed and swollen More than one toe, butnot entirepaw, inflamed and swollen Mild swelhnv ofentirepaw\ __________ aw1nflamedand swollen 4 Very inflamedandswollen paw or sed paw.“Ifthepaw 1 isankylosedthemouse cannotgrip thewiretopofthepage...“ At the onset of tis symptoms, mice were randomized into two groups and injected with the 252—3 antibody or polyclonal mulgG intraperitoneally at 10 mg/kg dose at three utive days. A total of 12 mice were enrolled for each group. At the end of the study (21 days after the e onset), the data were synchronized based on the day of disease onset for each mouse. The disease progression plot (Figure 11B) shows that the e severity in control mice sed y from mean score of 2 at day 1 to 9.5 at day 7. In contrast, the disease in mice treated with the 252—3 antibody progressed significantly slower with mean score of 4.4 at day 7. Altogether, this data suggests that the 252-2 antibody treatment significantly depletes the B cell population and alleviates CIA.

In sion, the above experiments using a surrogate anti—muCD37 antibody provide evidence that a CD37—targeting antibody, or an immunoconjugate that includes a CD37 antibody, can inhibit autoimmune diseases in animal models.

**** It is to be appreciated that the Detailed Description section, and not the Abstract section, is intended to be used to interpret the claims. The Abstract Abstract may set forth one or more but not all .. 9] .. exemplary embodiments of the present invention as contemplated by the inventors, and thus, is not intended to limit the present invention and the appended claims in any way.

The present invention has been described above with the aid of functional ng blocks illustrating the implementation of specified functions and relationships thereof The boundaries of these functional building blocks have been arbitrarily defined herein for the ience of the description.

Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately med.

The ing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for varéous applications such specific embodiments, without undue mentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of lents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be reted by the skilled artisan in light of the teachings and guidance.

] The breadth and scope of the present invention should not be limited by any of the above- described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All publications, s, and patent applications mentioned in this cation are herein incorporated by reference to the same extent as if each independent publication, patent, or patent application was cally and individually indicated to be incorporated by reference.

Claims (58)

What is claimed

1. Use of a ed humanized antibody or antigen-binding fragment thereof that ically binds to CD37 in the preparation of a medicament for ng a t having an mune or inflammatory e, wherein said humanized antibody or fragment thereof retains the ability of at least its chimeric or murine parent antibody to induce apoptosis in vitro in the absence of a cross-linking agent.

2. Use of a purified humanized antibody or n-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for depleting a B-cell in a population of cells comprising a non-cancerous B-cell, wherein said humanized antibody or fragment thereof retains the ability of at least its chimeric or murine parent antibody to induce apoptosis in vitro in the absence of a cross-linking agent.

3. The use according to claim 1, wherein the antibody is a resurfaced antibody.

4. The use ing to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to the same CD37 epitope as an antibody selected from the group consisting of: (a) an antibody comprising the polypeptide of SEQ ID NO:57 and the polypeptide of SEQ ID NO:74; (b) an antibody comprising the polypeptide of SEQ ID NO:63 and the polypeptide of SEQ ID NO:79; (c) an antibody comprising the polypeptide of SEQ ID NO:65 and the polypeptide of SEQ ID NO:81; (d) an antibody sing the polypeptide of SEQ ID NO:67 and the polypeptide of SEQ ID NO:83; (e) an antibody comprising the polypeptide of SEQ ID NO:69 and the polypeptide of SEQ ID NO:85; and (f) an antibody comprising the polypeptide of SEQ ID NO:71 and the polypeptide of SEQ ID NO:87.

5. The use according to claim 1, wherein the antibody or antigen-binding fragment thereof competitively inhibits the binding to CD37 of an antibody selected from the group consisting of: (a) an dy comprising the ptide of SEQ ID NO:57 and the polypeptide of SEQ ID NO:74; (b) an antibody sing the polypeptide of SEQ ID NO:63 and the polypeptide of SEQ ID NO:79; (c) an antibody comprising the polypeptide of SEQ ID NO:65 and the polypeptide of SEQ ID NO:81; (d) an antibody sing the polypeptide of SEQ ID NO:67 and the polypeptide of SEQ ID NO:83; (e) an antibody comprising the polypeptide of SEQ ID NO:69 and the polypeptide of SEQ ID NO:85; and (f) an antibody comprising the polypeptide of SEQ ID NO:71 and the polypeptide of SEQ ID NO:87.

6. The use according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to human CD37 and macaque CD37.

7. The use according to claim 2, wherein the dy or antigen-binding fragment thereof specifically binds to human CD37 and macaque CD37.

8. The use according to claim 6, wherein the antibody or n-binding nt thereof is an antibody produced by a hybridoma selected from the group consisting ATCC Deposit Designation PTA-10665, ted with the ATCC on February 18, 2010, ATCC t Designation PTA-10666, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10667 deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10668, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10669, deposited with the ATCC on February 18, 2010, and ATCC Deposit Designation PTA-10670, ted with the ATCC on February 18, 2010 or is an antigen-binding fragment thereof.

9. The use according to claim 7, wherein the antibody or antigen-binding fragment is an antibody produced by a hybridoma selected from the group consisting of ATCC Deposit Designation PTA-10665, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10666, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation 667 deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10668, deposited with the ATCC on February 18, 2010, ATCC Deposit Designation PTA-10669, deposited with the ATCC on February 18, 2010, and ATCC Deposit Designation PTA-10670, deposited with the ATCC on February 18, 2010 or is an antigen binding fragment thereof.

10. The use according to claim 1, wherein said antibody comprises polypeptide sequences selected from the group consisting of: (a) SEQ ID NOs: 4, 5, and 6 and SEQ ID NOs: 28, 29, and 30; (b) SEQ ID NOs: 10, 11, and 12 and SEQ ID NOs: 34, 35, and 36; (c) SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 37, 40, and 39; (d) SEQ ID NOs: 16, 17, and 18 and SEQ ID NOs: 41, 42, and 43; (e) SEQ ID NOs: 19, 20, and 21 and SEQ ID NOs: 44, 47, and 46; (f) SEQ ID NOs: 22, 23, and 24 and SEQ ID NOs: 48, 51, and 50; and (g) SEQ ID NOs: 25, 26, and 27 and SEQ ID NOs: 52, 53, and 54.

11. The use according to claim 1, wherein the antibody or antigen-binding nt thereof comprises polypeptide sequences that are at least 90%, 95%, or 99% cal to polypeptide sequences selected from the group consisting of: (a) SEQ ID NO:57 and SEQ ID NO:74; (b) SEQ ID NO:58 and SEQ ID NO:74; (c) SEQ ID NO:63 and SEQ ID NO:79; (d) SEQ ID NO:65 and SEQ ID NO:81; (e) SEQ ID NO:67 and SEQ ID NO:83; (f) SEQ ID NO:69 and SEQ ID NO:85; and (g) SEQ ID NO:71 and SEQ ID NO:87.

12. Use of a ed humanized antibody or antigen-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for treating a patient having an autoimmune or inflammatory e, wherein the antibody or antigen-binding fragment thereof ses polypeptide sequences selected from the group consisting of: (a) SEQ ID NO:57 and SEQ ID NO:74; (b) SEQ ID NO:58 and SEQ ID NO:74; (c) SEQ ID NO:63 and SEQ ID NO:79; (d) SEQ ID NO:65 and SEQ ID NO:81; (e) SEQ ID NO:67 and SEQ ID NO:83; (f) SEQ ID NO:69 and SEQ ID NO:85; and (g) SEQ ID NO:71 and SEQ ID NO:87.

13. Use of a purified humanized antibody or n-binding fragment thereof that specifically binds to CD37 in the preparation of a medicament for ing a B-cell in a population of cells comprising a non-cancerous B-cell, wherein the antibody or antigen-binding fragment thereof comprises polypeptide sequences selected from the group consisting of: (a) SEQ ID NO:57 and SEQ ID NO:74; (b) SEQ ID NO:58 and SEQ ID NO:74; (c) SEQ ID NO:63 and SEQ ID NO:79; (d) SEQ ID NO:65 and SEQ ID NO:81; (e) SEQ ID NO:67 and SEQ ID NO:83; (f) SEQ ID NO:69 and SEQ ID NO:85; and (g) SEQ ID NO:71 and SEQ ID NO:87.

14. The use according to claim 1, wherein said humanized antibody or antigen binding fragment thereof is also capable of inducing antibody dependent cell mediated cytotoxicity

15. The use according to any one of claims 1-14, which is a full length antibody.

16. The use according to any one of claims 1-14, which is an n-binding nt.

17. The use according to any one of claims 1-14, wherein said antibody or antigen-binding fragment thereof comprises a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR , IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, ody, dy, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

18. The use according to any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof is linked via a linker (L) to a cytotoxic agent (C) to form an immunoconjugate.

19. The use according to claim 18, wherein the immunoconjugate further comprises a second (C).

20. The use according to claim 18, wherein the immunoconjugate further comprises a third (C).

21. The use according to claim 18, wherein the immunoconjugate further comprises a fourth (C).

22. The use according to claim 18, wherein the immunoconjugate comprises two to six (C).

23. The use according to claim 18, wherein the immunoconjugate comprises three to four (C).

24. The use ing to claim 18, wherein said linker is selected from the group ting of a ble linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid based linker.

25. The use according to claim 24, wherein said linker is selected from the group consisting of: inimidyl 4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl 4-(2- pyridyldithio)butanoate (SPDB) or N-succinimidyl 4-(2-pyridyldithio)sulfobutanoate (sulfo-SPDB); N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N- sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N- succinimidyl(iodoacetyl)-aminobenzoate (SIAB); and N-succinimidyl-[(N- maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide).

26. The use according to claim 25, wherein said linker is SMCC.

27. The use according to claim 18, wherein said cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, doxorubicin, a modified doxorubicin, benzodiazepine, , CC-1065, CC-1065 analog, duocarmycin, duocarmycin , calicheamicin, dolastatin, dolastatin analog, auristatin, tomaymycin derivative, and leptomycin derivative or a prodrug of the agent.

28. The use according to claim 27, wherein said cytotoxic agent is a maytansinoid.

29. The use according to claim 28, n said xic agent is N(2')-deacetyl-N(2')-(3- mercaptooxopropyl)-maytansine (DM1) or N(2')-deacetyl-N(2')-(4-mercaptomethyl- entyl)-maytansine (DM4).

30. The use according to claim 29, wherein the immunoconjugate comprises 3 or 4 (C) per (A).

31. The use according to claim 18, wherein the antibody or antigen-binding fragment comprises the polypeptides of SEQ ID NOs: 4, 5, and 6 and SEQ ID NOs: 28, 29, and 30.

32. The use according to claim 31, wherein (L) is SMCC.

33. The use according to claim 31, wherein (C) is DM1 or DM4.

34. The use according to claim 32, wherein (C) is DM1 or DM4.

35. The use ing to claim 34, wherein (C) is DM1.

36. The use according to claim 35, wherein the immunoconjugate comprises 3 or 4 (C) per (A).

37. The use according to claim 18, n the antibody or antigen-binding fragment comprises the polypeptide of SEQ ID NO:57 and the polypeptide of SEQ ID NO:74.

38. The use according to claim 37, wherein (L) is SMCC.

39. The use ing to claim 37, wherein (C) is DM1 or DM4.

40. The use ing to claim 38, wherein (C) is DM1 or DM4.

41. The use according to claim 40, wherein (C) is DM1.

42. The use according to claim 41, wherein the immunoconjugate comprises 3 or 4 (C) per (A).

43. The use according to claim 18, wherein the dy or antigen-binding fragment comprises the polypeptide of SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 37, 40, and 39.

44. The use according to claim 43, wherein (L) is SMCC.

45. The use according to claim 43, wherein (C) is DM1 or DM4.

46. The use according to claim 44, wherein (C) is DM1 or DM4.

47. The use ing to claim 46, wherein (C) is DM1.

48. The use according to claim 47, wherein the immunoconjugate ses 3 or 4 (C) per (A).

49. The use according to claim 18, wherein the antibody or antigen-binding fragment comprises the polypeptide of SEQ ID NO:65 and the polypeptide of SEQ ID NO:81.

50. The use according to claim 49, wherein (L) is SMCC.

51. The use according to claim 49, wherein (C) is DM1 or DM4.

52. The use according to claim 50, wherein (C) is DM1 or DM4.

53. The use according to claim 52, wherein (C) is DM1.

54. The use according to claim 53, wherein the immunoconjugate comprises 3 or 4 (C) per (A).

55. The use according to claim 18, wherein said autoimmune or inflammatory disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type I diabetes mellitus, idiopathic inflammatory myopathy, systemic lupus erythematosus (SLE), myasthenia gravis, Grave's disease, dermatomyositis, polymyositis, Crohn's disease, ulcerative colitis, gastritis, oto's thyroiditis, asthma, psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sis, inflammatory bowel e (IBD), respiratory distress me, itis, encephalitis, uveitis, glomerulonephritis, eczema, atherosclerosis, leukocyte adhesion deficiency, Raynaud's syndrome, Sjögren's syndrome, Reiter's disease, 's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, atopic itis, pemphigus vulgaris, onus-myoclonus syndrome, pure red cell aplasia, mixed obulinemia, ankylosing spondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous goid, hemophilia A, membranoproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary sis, neuromyelitis optica, Graves' dysthyroid disease, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss syndrome, juvenile onset diabetes, hemolytic anemia, atopic dermatitis, systemic sis, Sjögren's syndrome and glomerulonephritis, dermatomyositis, anti-neutrophil cytoplasmic antibody (ANCA), aplastic , autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, mune neutropenia, Castleman's me, Goodpasture's syndrome, solid organ transplant rejection, graft versus host disease (GVHD), autoimmune hepatitis, lymphoid interstitial pneumonitis, HIV, bronchiolitis obliterans (non-transplant), Guillain-Barre Syndrome, large vessel vasculitis, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's Disease, polyarteritis nodosa, Wegener's granulomatosis, microscopic polyangiitis (MPA), Omenn's syndrome, c renal failure, acute infectious mononucleosis, HIV and herpes virus associated diseases.

56. The use according to claim 55, wherein said mune or matory e is multiple sclerosis.

57. The use according to claim 55, wherein said autoimmune or inflammatory disease is diabetes mellitus.

58. The use according to claim 55, wherein said autoimmune or inflammatory disease is rheumatoid arthritis. ImmunoGen, Inc. By the patent attorneys for the applicant CULLENS